biomolecules

Review JAK-Inhibitors for the Treatment of : A Focus on the Present and an Outlook on the Future

1, 2, , 3 1,4 Jacopo Angelini † , Rossella Talotta * † , Rossana Roncato , Giulia Fornasier , Giorgia Barbiero 1, Lisa Dal Cin 1, Serena Brancati 1 and Francesco Scaglione 5

1 Postgraduate School of Clinical and Toxicology, University of Milan, 20133 Milan, Italy; [email protected] (J.A.); [email protected] (G.F.); [email protected] (G.B.); [email protected] (L.D.C.); [email protected] (S.B.) 2 Department of Clinical and Experimental Medicine, Unit, AOU “Gaetano Martino”, University of Messina, 98100 Messina, Italy 3 Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico di Aviano (CRO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Pordenone, 33081 Aviano, Italy; [email protected] 4 Pharmacy Unit, IRCCS-Burlo Garofolo di Trieste, 34137 Trieste, Italy 5 Head of Clinical Pharmacology and Toxicology Unit, Grande Ospedale Metropolitano Niguarda, Department of Oncology and Onco-Hematology, Director of Postgraduate School of Clinical Pharmacology and Toxicology, University of Milan, 20162 Milan, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-090-2111; Fax: +39-090-293-5162 Co-first authors. †  Received: 16 May 2020; Accepted: 1 July 2020; Published: 5 July 2020 

Abstract: inhibitors (JAKi) belong to a new class of oral targeted disease-modifying drugs which have recently revolutionized the therapeutic panorama of rheumatoid arthritis (RA) and other immune-mediated diseases, placing alongside or even replacing conventional and biological drugs. JAKi are characterized by a novel mechanism of action, consisting of the intracellular interruption of the JAK-STAT pathway crucially involved in the immune response. The aim of this narrative review is to globally report the most relevant pharmacological features and clinical outcomes of the developed and incoming JAKi for RA, based on the available preclinical and clinical evidence. A total of 219 papers, including narrative and systematic reviews, randomized controlled trials (RCTs), observational studies, case reports, guidelines, and drug factsheets, were selected. The efficacy and safety profile of both the first generation JAKi ( and ) and the second generation JAKi (, filgotinib, peficitinib, and itacitinib) were compared and discussed. Results from RCTs and real-life data are encouraging and outline a rapid onset of the pharmacologic effects, which are maintained during the time. Their efficacy and safety profile are comparable or superior to those of biologic agents and JAKi proved to be efficacious when given as monotherapy. Finally, the manufacturing of JAKi is relatively easier and cheaper than that of biologics, thus increasing the number of compounds being formulated and tested for clinical use.

Keywords: Janus kinases; Janus kinase-inhibitors; rheumatoid arthritis; small molecules

1. Introduction Rheumatoid arthritis (RA) is a chronic affecting approximately 0.5–1% of the worldwide population. RA prevalence is higher in women aged between 35 and 50 years than in age-matched men, though this difference is less evident among elderly patients [1,2].

Biomolecules 2020, 10, 1002; doi:10.3390/biom10071002 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 1002 2 of 40

RA is characterized by a chronic synovitis that symmetrically develops in small joints of hands and feet, but any synovial joint can actually be involved [3]. Articular manifestations include swelling, tenderness, warmth, and decreased range of motion. Over time, persistent inflammation leads to the destruction of joints and tendons, and, eventually, to deformities and ankylosis. In some cases, RA can have an extra-articular presentation, and inflammation may involve the skin, heart, lungs, and eyes [4,5]. Generalized malaise and fatigue, pleural involvement, , pericarditis, myocardial infarction, rheumatoid nodules, nerve entrapment syndromes, and keratoconjunctivitis sicca are the most common extra-articular manifestations [6]. The etiology of RA is multifactorial, and the initial cause is unknown. It is assumed that in a genetically predisposed individual, an environmental agent, like or dysbiosis, can trigger an aberrant immune response against self-antigens placed in the articular sites [7,8]. Genetic predisposition is mirrored by the high concordance rates in twins, and several cases have been shown to occur in the same family [9]. More than one hundred genetic loci have been associated with RA risk [10], most of which preside over the control of the immune response [11,12]. Among them, polymorphic variants of the human leukocyte antigen (HLA) genes, coding for molecules involved in the antigen presentation process, have been associated to a more aggressive course of the disease or higher mortality rates [10,13]. Smoking, traumatic events, a low socioeconomic status, educational attainment, and periodontal disease are instead considered environmental risk factors for RA [14,15]. RA may be triggered by gut and oral dysbiosis or by infections sustained by Proteus mirabilis, Escherichia coli, or the Epstein–Barr [16,17]. Epigenetics may play an additional role in RA pathogenesis, being in turn influenced by environmental stimuli [18]. Histone deacetylation, DNA methylation, and microRNA production may affect the transcription of genes involved in inflammation, and are associated with disease risk and activity and response to treatment [19]. RA inflammation is due to the clonal expansion of autoreactive T cells, such as T helper (Th)17 and B lymphocytes at the detriment of T regulatory(reg) lymphocytes [20,21]. B lymphocytes mature to the final stage of plasma cells producing autoantibodies, including anti-citrullinated peptide (ACPAs) and rheumatoid factor (RF) that represent the serologic hallmark of the disease [21]. Autoreactive cells are recruited in the synovial membrane, where resident fibroblast-like and -like synoviocytes further contribute to the amplification of the inflammatory cascade through the release of several pro-inflammatory [7], like interleukin (IL)-1 and -alpha (TNF-α), IL-6, and IL-8 [22]. Additionally, Th17 lymphocytes secrete IL-17 that is crucially involved in bone resorption [20]. Persistent inflammation results in profound changes of the joint anatomy and physiology, progressing from synovial hyperplasia and endothelial cell activation in the early phase to cartilage destruction and bone erosion in the late phase [23]. The recent 2020 European League Against Rheumatism (EULAR) and the 2015 American College of Rheumatology (ACR) guidelines recommended to start treating RA patients as early as possible owing to the debilitating course of the disease, and subjects should be tightly monitored as therapy should be adjusted according to disease activity [24,25]. The treatment of RA relies on the use of drugs counteracting the aberrant activation of the and includes anti-inflammatory and analgesic drugs, glucocorticoids, disease-modifying anti-rheumatic drugs (DMARDs), immunosuppressive agents, and biologics. The DMARD (MTX), and to a lesser extent, leflunomide and , have been considered as an anchor therapy in RA. Accordingly, their use, as mono- or combo-therapy, is recommended in the early phase of the disease, as well as to treat the milder forms of RA in the long term. Glucocorticoids and non-steroidal anti-inflammatory drugs (NSAIDs) are, instead, indicated for the acute management of RA flares, while the chronic use of these drugs is discouraged. Conventional (c)DMARDs, glucocorticoids, and NSAIDs are characterized by a low target specificity and may unselectively hamper physiological Biomolecules 2020, 10, 1002 3 of 40 pathways other than the immune response, exposing patients to a not negligible risk of adverse events, like infections or gastrointestinal, cardiovascular, and hematologic disorders [26–28]. Since their advent in late 1990s [29], biological drugs consisting of monoclonal antibodies or fusion receptors targeting specific molecular or cellular pathways, notably improved the clinical course of RA, allowing the achievement of low disease activity or even remission in a high percentage of cases [30,31]. Additionally, in the last years, the formulation of oral compounds, known as small molecules, able to block some crucial steps of the inflammatory cascade, has further enriched RA therapeutic armamentarium. According to current therapeutic guidelines [24,25], biologic drugs and synthetic small molecules should be prescribed in the case of severe and refractory RA, but these may also be considered as the first therapy when poor prognostic factors are present [24]. Among synthetic small molecules, the Janus kinase inhibitors (JAKi) represent a new class of oral drugs counteracting the activation of JAKs, which are cytosolic presiding over many biologic functions, including the activation of the inflammatory cascade in immune cells [32]. Due to their central role in the immune response and their association with several receptors, the inhibition of JAKs appeared to be a promising strategy in autoimmune diseases. To date, some oral JAKi (tofacitinib, baricitinib, upadacitinib, peficitinib) have already been licensed for the treatment of RA and other immune-mediated diseases. Results from randomized controlled trials (RCTs) and real-life data are encouraging, and evidence a rapid onset of the pharmacologic effects, which are maintained over the course of time. Their efficacy and safety profile are comparable or superior to those of biologic agents, and JAKi proved to be efficacious when given as monotherapy [33]. In addition, the manufacturing of JAKi is relatively easier and cheaper than that of biologics, and thus noteworthy for increasing the number of compounds being formulated and tested for clinical use. The panorama of JAKi designed for RA is extremely innovative and dynamic. The aim of this narrative review is therefore to comprehensively report and compare the pharmacological profile of JAKi approved for the treatment of RA, also providing evidence on JAKi currently under development. We discuss data concerning mechanistic, clinical, and pharmacoeconomic aspects, in order to support clinicians in the identification of the most appropriate place in therapy of these promising new drugs.

2. Methods A literature research was conducted using PubMed, ClinicalTrials.gov, and Google Scholar databases, searching for the medical subject headings (MeSH) terms “rheumatoid arthritis”, “JAK-inhibitors”, “baricitinib”, “tofacitinib”, “upadacitinib”, “peficitinib”, “filgotinib”, “decernotinib”, and “itacitinib”. A total of 219 papers, written in English and published between 1994 and 2020 and pertinent to the purpose of the review, were selected. They included narrative reviews (n. 74), RCTs (n. 70), systematic and evidence reviews and meta-analyses (n. 11), retrospective cohort studies (n. 16), observational case control studies (n. 31), cross-sectional and interventional studies (n. 1 and n. 2, respectively), case reports (n. 1), guidelines (n. 3), and drug factsheets and reports (n. 10) (Figure1). Results are presented and discussed in the next sections. Biomolecules 2020, 10, 1002 4 of 40 Biomolecules 2019, 9, x FOR PEER REVIEW 4 of 38

Figure 1. Literature selection process for this article (JAKi, JAK-inhibitors; RCTs, randomized controlled trials). Figure 1. Literature selection process for this article (JAKi, JAK-inhibitors; RCTs, randomized 3. Generalcontrolled Pharmacological trials). Properties of JAKi Cytokine signaling has been considered as an optimal pharmacological target for the treatment of 3. General Pharmacological Properties of JAKi RA and other autoimmune diseases. Both conventional and biological drugs approved for RA, in fact, act byCytokine preventing signaling cytokine has release been considered and signaling. as an optimal pharmacological target for the treatment of RAJAKi and block other the autoimmune specific adenosine diseases. triphosphate Both conventional (ATP)-binding and biological pocket, interruptingdrugs approved the JAK-Signal for RA, in Transducerfact, act by preventing and Activator cytokine of Transcription release and (STAT) signaling. intracellular signaling cascade that ultimately leads to theJAKi activation block of the immune specific cells adenosine and their triphosphate transition towards (ATP) a-binding pro-inflammatory pocket, interrupting phenotype [34the]. FourJAK-Signal different Transducer types of JAKsand A (JAK1,ctivator JAK2, of Transcription JAK3, and tyrosine (STAT) kinaseintracellular 2, TYK2) signaling and seven cascade different that STATsultimately (STAT1, leads STAT2, to the STAT3, activation STAT4, of immune STAT5A, cells STAT5B, and their STAT6) transition can variously towards combine a pro- andinflammatory give raise tophenotype different [34] biologic. Four cascades different [35types], Table of JAKs1. JAKs (JAK1, are JAK2, enzymes JAK3 belonging, and tyrosine to the kinase family 2, ofTYK2) tyrosine and kinasesseven different constitutively STATs bound (STAT1, to the STAT2, intracellular STAT3, domains STAT4, ofSTAT5A, type I and STAT5B, II receptors. STAT6) Type can I variouslyreceptors bindcombine several and ILs,give colony raise to stimulating different biologic factors, cascades and hormones [35], Table such 1. as JAKs , are enzymes prolactin, belonging and to growththe family hormone. of tyrosine Type kinasesII receptors constitutively bind bound and to IL-10 the intracellular related cytokines domains [36]. of type I and II receptors.In the Type canonical I rece pathway,ptors bind the interactionseveral ILs, between colony cytokines stimulating and factors their, cognate and hormones receptors such induce as theirerythropoietin, dimerization prolactin and the, and downstream growth hormone. activation Type of JAKs. II receptors Generally, bind cytokine interferons receptors and IL do-10 not related have intrinsiccytokines kinase [36]. activity, and use the binding of JAK enzymes for the autophosphorylation of tyrosine residuesIn the present canonical in the pathway, cytoplasmic the interaction domains. between This phosphorylation cytokines and their leads cognate to the dockingreceptors and induce the activationtheir dimerization of the STAT and proteins. the downstream JAKs phosphorylate activation STAT of JAKs. proteins Generally, with the cytokine consequent receptors dimerization do not ofhave STAT int monomersrinsic kinase and activity, translocation and use into the the binding nucleus. of Here,JAK enzymes STAT molecules for the behaveautophosphorylation as transcriptional of factors,tyrosine promoting residues present the transcription in the cytoplasmic of target genes domains. [37], asThis shown phosphorylation in Figure2. leads to the docking and the activation of the STAT proteins. JAKs phosphorylate STAT proteins with the consequent dimerization of STAT monomers and translocation into the nucleus. Here, STAT molecules behave as transcriptional factors, promoting the transcription of target genes [37], as shown in Figure 2.

Table 1. Combinations of cytokines, types of JAK -STAT proteins, cellular targets and possible related disorders.

Type of Associated Associated Diseases Linked to an Altered Cytokine JAK STAT Main Target Organ/Cell References Pathway Receptor Subtype Subtype alopecia, rheumatoid arthritis, STAT3 JAK1 naïve T lymphocytes, Th1 systemic erythematosus, IL-2 Type I STAT5A/B [38–43] JAK3 lymphocytes , Crohn’s disease,

STAT3 alopecia, rheumatoid arthritis, IL-3 Type I JAK2 STAT5A/B Th2 lymphocytes atopic , systemic lupus [38,39,44] STAT6 erythematosus, IL-4 Type I JAK1 STAT6 naïve T lymphocytes, Th2 alopecia, rheumatoid arthritis, [38,39,45] Biomolecules 2020, 10, 1002 5 of 40

Table 1. Combinations of cytokines, types of JAK receptor-STAT proteins, cellular targets and possible related disorders.

Type of Associated Associated Diseases Linked to an Ligand Cytokine JAK STAT Main Target Organ/Cell References Altered Pathway Receptor Subtype Subtype alopecia, rheumatoid arthritis, systemic lupus JAK1 STAT3 naïve T lymphocytes, Th1 IL-2 Type I erythematosus, psoriasis, [38–43] JAK3 STAT5A/B lymphocytes Crohn’s disease, ankylosing spondylitis alopecia, rheumatoid arthritis, STAT3 , systemic IL-3 Type I JAK2 STAT5A/B Th2 lymphocytes [38,39,44] , STAT6 ulcerative colitis alopecia, rheumatoid arthritis, atopic dermatitis, systemic JAK1 naïve T lymphocytes, Th2 IL-4 Type I STAT6 lupus erythematosus, [38,39,45] JAK3 lymphocytes psoriasis, ankylosing spondylitis, ulcerative colitis alopecia, rheumatoid arthritis, STAT3 atopic dermatitis, systemic IL-5 Type I JAK2 STAT5A/B Th2 lymphocytes [38,39,44] lupus erythematosus, STAT6 ulcerative colitis alopecia, rheumatoid arthritis, atopic dermatitis, systemic JAK1 STAT1 naïve T lymphocytes, lupus erythematosus, IL-6 Type I JAK2 [11,38,39,46,47] STAT3 human keratinocytes ulcerative colitis, Crohn’s TYK2 disease, ankylosing spondylitis, psoriasis alopecia, rheumatoid arthritis, systemic lupus JAK1 STAT3 naïve T lymphocytes, Th1 IL-7 Type I erythematosus, psoriasis, [38–43] JAK3 STAT5A/B lymphocytes Crohn’s disease, ankylosing spondylitis alopecia, rheumatoid arthritis, systemic lupus JAK1 STAT3 naïve T lymphocytes, Th1 IL-9 Type I erythematosus, psoriasis, [38–43] JAK3 STAT5A/B lymphocytes Crohn’s disease, ankylosing spondylitis JAK1 alopecia, rheumatoid STAT3 IL-10 Type II JAK2 Treg lymphocytes arthritis, systemic lupus [38,39,48] STAT5A/B TYK2 erythematosus alopecia, rheumatoid naïve T lymphocytes, arthritis, atopic dermatitis, JAK1 immunoglobulin-producing systemic lupus STAT1 IL-11 Type I JAK2 B cells, hematopoietic stem erythematosus, ulcerative [11,38,39,46,47] STAT3 TYK2 cells, megakaryocyte colitis, Crohn’s disease, progenitor cells ankylosing spondylitis, , alopecia, rheumatoid arthritis, atopic dermatitis, JAK2 systemic lupus IL-12 Type I STAT4 naïve T lymphocytes [11,38,39,49] TYK2 erythematosus, ulcerative colitis, Crohn’s disease, ankylosing spondylitis JAK1 alopecia, rheumatoid arthritis, Th2 lymphocytes, human JAK2 atopic dermatitis, systemic IL-13 Type I STAT6 bronchial smooth muscle [38,39,50] JAK3 lupus erythematosus, cells TYK2 ulcerative colitis, asthma alopecia, rheumatoid arthritis, systemic lupus JAK1 STAT3 naïve T lymphocytes, Th1 IL-15 Type I erythematosus, psoriasis, [38–43] JAK3 STAT5A/B lymphocytes Crohn’s disease, ankylosing spondylitis JAK1 IL-19 Type II JAK2 STAT3 innate immune system / [38,39,51] TYK2 JAK1 innate immune system, IL-20 Type II JAK2 STAT3 psoriasis [38,39,52,53] human keratinocytes TYK2 alopecia, rheumatoid arthritis, systemic lupus JAK1 STAT3 naïve T lymphocytes, Th1 IL-21 Type I erythematosus, psoriasis, [38–43,54] JAK3 STAT5A/B lymphocytes Crohn’s disease, ankylosing spondylitis Biomolecules 2020, 10, 1002 6 of 40

Table 1. Cont.

Type of Associated Associated Diseases Linked to an Ligand Cytokine JAK STAT Main Target Organ/Cell References Altered Pathway Receptor Subtype Subtype alopecia, rheumatoid JAK1 STAT1 arthritis, systemic lupus IL-22 Type II JAK2 STAT3 Th17 lymphocytes erythematosus, Crohn’s [38,39,53] TYK2 STAT5A/B disease, ankylosing spondylitis, psoriasis alopecia, rheumatoid arthritis, atopic dermatitis, TYK2 STAT3 systemic lupus IL-23 Type I naïve T lymphocytes [11,38,39,49,55] JAK2 STAT4 erythematosus, ulcerative colitis, Crohn’s disease, ankylosing spondylitis immune cells, colonic IL-24 Type II JAK1 STAT3 inflammatory bowel disease [38,39,56] epithelial cells STAT1 JAK1 STAT2 Th1 lymphocytes, cytotoxic IL-27 Type I JAK2 STAT3 autoimmune disorders [38,39,57] , Treg lymphocytes TYK2 STAT4 STAT5A/B STAT1 STAT2 JAK1 immune cells, human IL-28 Type II STAT3 / [38,39,54] TYK2 keratinocytes STAT4 STAT5A/B STAT1 STAT2 JAK1 IL-29 Type II STAT3 immune cells / [38,39,54] TYK2 STAT4 STAT5A/B STAT1 JAK1 lung, skin, thymus, spleen, IL-31 Type I STAT3 atopic dermatitis [38,58] JAK2 myelomonocytic cells STAT5A/B alopecia, rheumatoid STAT1 arthritis, atopic dermatitis, JAK1 STAT2 naïve T lymphocytes, Th1 systemic lupus IFN-α/β Type II [11,38,39,59,60] TYK2 STAT4 lymphocytes erythematosus, ulcerative STAT3 colitis, Crohn’s disease, ankylosing spondylitis alopecia, rheumatoid arthritis, naïve T lymphocytes, Th1 atopic dermatitis, systemic JAK1 lymphocytes, human lupus erythematosus, IFN-γ Type II STAT1 [38,39,61] TYK2 salivary glands, human ulcerative colitis, Crohn’s keratinocytes disease, ankylosing spondylitis, psoriasis cells belonging to the lineage, from anemia, leukopenia, GM-CSF and STAT3 Type I JAK2 haemopoietic stem cells to autoimmune disorders, acute [38,39,62,63] G-CSF STAT5A/B mature , antigen myelogenous presenting cells STAT3 GH Type I JAK2 all tissues / [38,39,50,64,65] STAT5A/B erythroid precursor cell at EPO Type I JAK2 STAT5A/B anemia [38,39,66] colony-forming units STAT1 earliest erythroid Type I JAK2 STAT3 [38,39,67] progenitors STAT5A/B STAT3 Type I JAK2 brain, peripheral tissues / [38,39,68] STAT5A/B (JAK: Janus kinase; STAT: JAK signal transducer and activator of transcription; IL: interleukin; GM-CSF: granulocyte-macrophage colony-stimulating factor; TYK: ; Th: T helper; GH: growth hormone; EPO: erythropoietin; G-CSF: granulocyte-colony stimulating factor; IFN: ; Treg, T regulatory). Biomolecules 2019, 9, x FOR PEER REVIEW 6 of 38

Crohn’s disease, ankylosing spondylitis, psoriasis cells belonging to the neutrophil anemia, leukopenia, autoimmune GM-CSF and STAT3 lineage, from haemopoietic stem cells Type I JAK2 disorders, acute myelogenous [38,39,62,63] G-CSF STAT5A/B to mature neutrophils, antigen leukemia presenting cells STAT3 GH Type I JAK2 all tissues / [38,39,50,64,65] STAT5A/B erythroid precursor cell at EPO Type I JAK2 STAT5A/B anemia [38,39,66] colony-forming units STAT1 Thrombopoietin Type I JAK2 STAT3 earliest erythroid progenitors thrombocytopenia [38,39,67] STAT5A/B STAT3 Leptin Type I JAK2 brain, peripheral tissues / [38,39,68] STAT5A/B (JAK: Janus kinase; STAT: JAK signal transducer and activator of transcription; IL: interleukin; GM-CSF: granulocyte-macrophage colony-stimulating factor; TYK: tyrosine kinase; Th: T helper; GH: growth hormone; EPO: erythropoietin; G-CSF: granulocyte-colony stimulating factor; IFN: Biomolecules 2020, 10, 1002 7 of 40 interferon; Treg, T regulatory).

Figure 2. The JAK-STAT canonical signaling pathway. The specific cytokine binds to the transmembrane

receptors type I o II. This binding causes the auto-phosphorylation of the receptor itself which summons cytosolicFigure monomeric 2. The JAK JAK-STAT proteins canonical (1). Once signaling recruited, pathway. JAKs phosphorylate The specific the (2 binds), allowing, to the intransmembrane turn, a second phosphorylation receptors type I of o STATII. This monomers binding (causes3,4). STATs the auto dimerize-phosphorylation (5) and translocate of the intoreceptor the nucleusitself which (6), activating summons the cytosolic transcription monomeric of pro-inflammatory JAK proteins (1 genes). Once (7 recruit). (JAK:ed, Janus JAKs kinase; phosphorylate STAT: JAK the signalreceptor transducer (2), allowing, and activator in turn, of a transcription; second phosphorylation IL: interleukin; of G-CSF:STAT monomers granulocyte-colony-stimulating (3,4). STATs dimerize factor;(5) and TYK: translocate tyrosine into kinase; the nucleus GH: growth (6), activating hormone; the EPO: transcription erythropoietin; of pro-inflammatory TPO: thrombopoietin; genes (7). IFN:(JAK: interferon). Janus kinase; STAT: JAK signal transducer and activator of transcription; IL: interleukin; G-CSF: granulocyte-colony-stimulating factor; TYK: tyrosine kinase; GH: growth hormone; EPO: Oferythropoietin; note, JAK1 and TPO: JAK2 thrombopoietin; are found ubiquitously IFN: interferon). and modulate the expression of many inflammatory and non-inflammatory genes in response to IL-6, IL-23, granulocyte colony-stimulating factor, interferons, Of note, erythropoietin JAK1 and and JAK2 other are ligands found [38 – ubiquit57,59–69ously]. JAK3 and is, instead,modulate expressed the expression in hematopoietic of many cellsinflammatory and is involved and in the non signaling-inflammatory cascades unleashed genes in by IL-2, response IL-4, IL-7, to IL-9,IL-6, IL-15, IL- and23, IL-21 granulocyte [34,36]. colonyThe-stimulating JAK-STAT pathway factor, interferons, is highly conserved erythropoietin among and species other due ligands to its [38 involvement–57,59–69].in JAK3 many is, physiologicalinstead, expressed processes, in hematopoietic including the cells antimicrobial and is involved response, in the , signaling cell cascades proliferation unleashed and by self-renew,IL-2, IL-4, andIL-7, tissue IL-9, IL regeneration-15, and IL- [2170 [34,36]]. The. kinase activity is strictly regulated by phosphoprotein phosphatasesThe JAK and-STAT ubiquitin pathway is highlythat dephosphorylate conserved among JAKs species or induce due the to proteasomal its involvement degradation in many ofphysiological the JAK-receptor proce complexsses, including [71]. Accordingly, the antimicrobial JAK and response, STAT loss-of-function metabolism, andcell gain-of-function proliferation and geneticself-renew variants, and have tissue been regeneration associated with[70]. immunodeficiencyThe kinase activity and is strictly growth regulated retardation by and phosphoprotein with andphosphatases , and respectively ubiquitin [72 ligases]. that dephosphorylate JAKs or induce the proteasomal degradationBesides the of canonical the JAK- pathway,receptor acomplex non-canonical [71]. Accordingly, JAK-STAT signaling JAK and mechanism STAT loss has-of- additionallyfunction and been described in animal models and human cells [73]. In Drosophila and mammalian cells, non-phosphorylated STAT forms may shuttle between cytosol and nucleus and affect the euchromatin/heterochromatin ratio without the engagement with STAT-activated genes [74]. Dimeric or multimeric STATs may form cytosolic molecular platforms recruiting chaperones or other proteins associated to organelles or involved in membrane trafficking [75]. Preclinical experiments showed that STAT3 may non-canonically preside over the integrity of microtubules and mitochondria and that STAT5A and STAT5B may control the normal functioning of the rough endoplasmic reticulum [76]. In nucleus, non-phosphorylated STAT1 and STAT3 molecules may couple with other transcriptional factors, like interferon regulatory factor-1 (IRF1) thus influencing the expression of additional genes [75]. Finally, JAK2 may epigenetically control gene transcription through histone phosphorylation [76]. It is worth underlining that the JAK-STAT pathway is not the only mechanism orchestrating the immune response in autoimmunity [77]. Other cytokines, like TNF-α, trigger, in fact, distinct intracellular cascades, mostly converging on the activation of the transcriptional factors nuclear factor Biomolecules 2020, 10, 1002 8 of 40 kappa--chain-enhancer of activated B cells (NF-kB) or nuclear factor of activated T-cells (NFAT), which eventually promote the expression of pro-inflammatory genes [78,79]. Notably, these signaling mechanisms may reciprocally influence one another: for instance, it has been shown that NF-kB may induce the expression of the suppressor of cytokine signaling (SOCS)3, in turn inactivating STAT3 in human glioblastoma cells [80], and that NFAT may engage with STAT3 in a dynamic ternary complex promoting the hypertrophy of cardiomyocytes in mouse models [81]. Furthermore, the evidence that JAK and STAT molecules may non-canonically modulate the cell transcriptome without requiring kinase activity should indeed deserve further investigation concerning a presumable residual activity during the pharmacologic inhibition of the JAK-STAT canonical pathway. According to their selectivity, JAKi can be divided into first generation JAKi, consisting of non-selective inhibitors, and second generation JAKi, inhibiting the signaling of a narrower range of cytokines. The first generation JAKi encompasses baricitinib, which inhibits JAK1 and JAK2, and tofacitinib, which inhibits JAK1, JAK2, JAK3 and, to a lesser extent, TYK2 [82]; the second generation JAKi, includes, instead, upadacitinib, decernotinib, filgotinib, peficitinib and itacitinib, most of which are still under development. Second generation JAKi seem to have a faster and dose-dependent efficacy and appear more appealing when used as mono-therapy [83]. More selective JAKi should have a better safety profile; however, the complex interplay among cytokines and the ubiquity of the JAK-STAT molecules in cells not belonging to the immune system, though being helpful in the treatment of a broader range of diseases, may increases the risk of unwanted side effects. Due to the repression of the immune response, infections, especially of the upper respiratory tract, are the most common side effect during the treatment with JAKi. In addition, reactivation of Herpes Zoster virus (HZV) and alteration in the blood profile have typically been reported under JAKi therapy and appeared to be dose-dependent [84–86]. HZV reactivation seems to rely on the repression of the type I interferon response following JAK1 inhibition. Subsequently, vaccination against HZV is recommended before starting JAKi treatment [87,88], especially in some genetically-predisposed ethnic groups and in patients concomitantly prescribed with MTX [89]. JAKi may induce high density lipoprotein (HDL) efflux from or prevent the IL-6-induced storage of blood into peripheral tissues, and thus increase the level of low density lipoproteins (LDL), HDL and total [90], without affecting the LDL/HDL cholesterol ratio. Nevertheless, the increase in blood cholesterol levels has not been correlated to an augmented risk of cardiovascular disease in clinical trials, confirming the theory, also known as “the lipid paradox phenomenon” [91], that in RA cardiovascular morbidity and mortality mostly depend on chronic inflammation rather than on other classical risk factors [92]. Likewise, all clinical studies on JAKi have shown a low incidence of cardiovascular events in treated cohorts of patients probably related to the anti-inflammatory role played by these small molecules [93]. Thanks to the interesting efficacy profile emerging from phase III and long-term extension trials [94], it is expected that the use of JAKi for the treatment of RA will notably increase in the next years. This may be further supported by a better therapeutic compliance and a more favorable pharmacoeconomic impact than those of biological agents and their . The oral route of administration of JAKi has, in fact, the potential to minimize drug discontinuation in contrast to parentally administered biological products. Finally, the lower manufacturing costs of JAKi compared to those of biologics may result in a more positive pharmacoeconomic trend soon after the expiration of JAKi patent protection. In order to provide a comprehensive overview of the panorama of JAKi in RA, the pharmacologic aspects of marketed JAKi and those under development for RA are singularly delineated and discussed in the following paragraphs. Biomolecules 2020, 10, 1002 9 of 40

4. Baricitinib

4.1. Chemical Structure, , Pharmacodynamics and Mechanism of Action Baricitinib (International Union of Pure and Applied Chemistry (IUPAC) name: 2-[1-ethylsulfonyl-3-[4-(7H-pyrrolo[2 ,3-d]pyrimidin-4-yl)-1-pyrazolyl]-3-azetidinyl]acetonitrile) is a first generation JAKi, currently solely licensed for the treatment of moderate to severe active RA in adult patients who inadequately respond or are intolerant to one or more cDMARDs. Its chemical structure is that of a pyrrolopyrimidine, being insoluble in water and slightly soluble in hydrochloric acid [95,96]. Baricitinib was obtained by modifying the structure of tofacitinib, another first generation ATP-competitive JAKi. Both the drugs target the JH1 tyrosine kinase domain by interacting with the active conformational site of the ATP-binding pocket [97]. This structure is highly conserved among JAK enzymes, and, consequently, the first-generation JAKi unselectively target several JAKs. Baricitinib prevents the activation of both JAK1 and JAK2 molecules with half maximal inhibitory concentration (IC50) values of 5.9 and 5.7, respectively. This results in the inhibition of the expression of IL-6 in a dose-dependent manner [98]. When given to healthy subjects at a daily dose of 4 mg, the inhibition peaks after two hours since administration and lasts for 24 h [85]. Another relevant effect is the inhibition in vitro of osteoclastogenesis and thus subchondral bone erosions, through the down-regulation of receptor activator of nuclear factor-κB ligand (RANKL) in osteoblasts [99]. In adult patients aged <75 years and without known risk factors, baricitinib is orally administered at a dosage of 2 mg once daily. It can be used either in combination with MTX and other cDMARDs in patients partially responding to these medications or as monotherapy in those who discontinue conventional treatment due to intolerance or to the achievement of their treatment target [100]. Elderly subjects and those with a history of recurrent infections or renal function impairment should receive half-daily dose. Baricitinib pharmacokinetic profiles after single or multiple administrations are comparable in healthy subjects [101]. After absorption, the is 79% and, in blood, about 50% of the drug is bound to plasma proteins. In RA patients, the drug is rapidly absorbed with a time to peak (Tmax) of 1.5 h, and a mean half-life of 12.5 h [102]. Meals decrease the intestinal absorption by 14% and maximum serum concentration (Cmax) by 18–29%. The steady state is obtained within 48 h (or 6 half-life) × after the first dose. The mean is 76 L after intravenous (i.v.) administration. Baricitinib is metabolized by hepatic enzymes, mainly belonging to the cytochrome P450 cluster (CYP3A4). Accordingly, the co-administration of baricitinib with CYP3A4 inhibitors or inducers should be carefully evaluated case by case. Nevertheless, studies of clinical pharmacology suggest that baricitinib does not require any dose adjustment in case of mild or moderate hepatic impairment, co-administration of proton pump inhibitors (PPI), CYP3A4 inhibitors (e.g., ), moderate CYP3A/CYP2C19/CYP2C9 inhibitors (e.g., fluconazole) or strong CYP3A inducers (e.g., ) [85]. Furthermore, no meaningful interactions have been reported when other CYP3A drug substrates, such as MTX, simvastatin, ethinyl oestradiol, or levonorgestrel were co-administrated [103]. The drug is however contraindicated in patients with severe hepatic impairment [103]. Baricitinib is mostly eliminated by glomerular filtration and kidney tubule active transportation. This is mediated by organic anion transporter 3 (OAT3), glycoprotein-P, breast cancer resistance protein (BCRP), and multidrug and toxin extrusion protein 2 K (MATE-2K). Studies in RA patients with a preserved kidney function reported a renal clearance of baricitinib of 9.42 L/hour, which is slightly lower than that of healthy volunteers (12 L/hour) [101]. Patients with renal failure require a dose adjustment, and the drug is not recommended in patients having a creatinine clearance < 30 mL/minute [101,104]. Additionally, when baricitinib is co-administrated with digoxin, a substrate of glycoprotein-P, no meaningful alterations were detected [103]. Biomolecules 2020, 10, 1002 10 of 40

4.2. Efficacy The clinical efficacy of baricitinib as mono- or combo-therapy in patients with RA was assessed in a total of 4 randomized, double-blind, placebo-controlled phase III clinical trials: two long-term (52 weeks) active controlled trials (RA-BEGIN [105] and RA-BEAM Study [106]) and two shorter-term (24 weeks) 3-arm randomized placebo-controlled trials (RA-BUILD [107] and RA-BEACON [108]). Patients who completed one of these main phase III clinical trials or a phase II exploratory trial [109] were eligible to entering the ongoing phase III single-blind multicenter long-term extension study RA-BEYOND [110,111]. These studies recruited RA patients naïve to conventional and biologic drugs (RA-BEGIN), those failing a precedent treatment with at least one cDMARD (RA-BEAM, RA-BUILD), or those failing a previous biologic therapy, including an anti-TNF agent (RA-BEACON). Studies differed in design and statistical analysis. Specifically, the RA-BEGIN trial tested the non-inferiority of baricitinib vs. MTX, while RA-BEAM, RA-BUILD and RA-BEACON were superiority trials vs. placebo. All the registration trials achieved the primary endpoint, consisting of the percentage of patients meeting the ACR20 improvement criteria (ACR20) at week 12 or 24. In addition, all major secondary endpoints, including ACR50 and ACR70 response rates, disease activity score on 28 joints by C-reactive protein (CRP) (DAS28-CRP) response, health assessment questionnaire disability index (HAQ-DI) response, simplified disease activity index (SDAI) remission rate and improvement from baseline in patient reported outcomes (PROs), were reached in the baricitinib arm compared to placebo. Of note, in the RA-BEAM trial [106], baricitinib was head to head compared with the anti-TNF monoclonal , resulting in a significant superiority to adalimumab in ACR20, ACR50 and ACR70 response rate until week 52, and in DAS28-CRP scores, SDAI remission achievement, HAQ-DI scores, and several PROs [112] at week 12. In the RA-BUILD trial [107], a statistically significant reduction in the radiographic progression of structural joint damage from baseline to week 24 was observed for both 2 mg and 4 mg baricitinib groups compared with the placebo, though the effect was stronger with baricitinib 4 mg/day. These results were confirmed in a two-year analysis in patients who completed RA-BEGIN, RA-BEAM, and RA-BUILD trials [111]. The design of RA-BEYOND [110,111] included also a sub-study in which patients who received baricitinib 4 mg once daily for at least 15 months in originating studies and who achieved sustained low disease activity or remission were re-randomized to continue receiving baricitinib 4 mg once daily or stepping down to 2 mg daily with or without cDMARDs. This step-down sub-study was designed in accordance with international therapeutic guidelines, which recommend a dose tapering (but not discontinuation) of DMARDs in patients who have achieved a sustained disease control [25,113]. After 48 weeks, most of the patients assigned to either a step-down strategy or standard regimen were still in low disease activity or remission [114]. These results, together with some safety trends, including adverse events and rates that would seem to favor a 2 mg daily dose, suggest that a dose tapering strategy could be considered in those patients whose RA disease activity has been kept under control with an inductive standard regimen. However, compared with the 4 mg group, the reduction to 2 mg was associated with a modest but statistically significant increase in tender and swollen joint count, physician global assessment, DAS28-CRP, clinical disease activity index (CDAI), and SDAI scores. A synthesis of the main characteristics of the reported clinical trials on baricitinib is provided in Table2. Biomolecules 2020, 10, 1002 11 of 40

Table 2. Baricitinib phase III trials in moderate to severe rheumatoid arthritis.

RA-BEGIN MTX-Naïve RA-BEAM MTX-IR RA-BUILD cDMARD-IR RA-BEACON bDMARD-IR RA-BEYOND OLE Study Study (n = 588) (n = 1308) (n = 684) (n = 527) (n = 3073)

- Moderately to severe active RA - Patients must have previously received - Active RA and inadequate ≥ 1 TNFi and discontinued the treatment response or intolerance to - Active RA ≥ because of an inadequate response or -RA 1 cDMARD - Patients with inadequate unacceptable side effects - Patients who - Patients who received no - Use of up to 2 concomitant response to MTX, who received - bDMARDs must have been discontinued completed a BARI prior cDMARD therapy (up cDMARDs was permitted at Inclusion criteria therapy for 12 weeks before trial at least 4 weeks before randomization phase II or phase to 3 weekly MTX ≥ entry; these must have been entry, including 8 weeks at ( 6 months for ) III trial doses permitted) ≥ used for at least 12 preceding ≥ stable doses - Use of 1 concomitant cDMARD at weeks with stable doses for at ≥ entry; these must have been used for at least 8 preceding weeks least 12 preceding weeks with stable doses for at least 8 preceding weeks

Monotherapy—patients Monotherapy + combination Type of therapy Combination therapy Combination therapy who completed previous therapy BARI RA studies Background None/MTX MTX cDMARDs cDMARDs cDMARDs treatment Active comparator MTX ADA + MTX (1) BARI 4 mg sid (1) PBO (1) BARI 2 mg sid (1) BARI 2 mg sid (1) BARI 2 mg sid Arms (2) BARI 4 mg sid + MTX (2) BARI 4 mg sid (2) BARI 4 mg sid (2) BARI 4 mg sid (2) BARI 4 mg sid (3) MTX 10 mg/week (3) ADA 40 mg/sc q2wk (3) PBO (3) PBO Ongoing (completion Duration (weeks) 52 52 24 24 estimated in 2024) ACR20 ACR20 ACR20 ACR20 Primary endpoint Long term Safety (Week 24) (Week 12) (Week 12) (Week 12) Week 12: Week 24: Week 12: DAS28-CRP Week 12: DAS28-CRP DAS28-CRP Key secondary HAQ-DI DAS28-CRP HAQ-DI HAQ-DI Long term Efficacy endpoint mTSS (Week 24) HAQ-DI mTSS SDAI remission SDAI remission SDAI remission SDAI remission Morning Joint stiffness Morning Joint stiffness (Week 24) BARI 4 mg vs. MTX: 77% vs. 62% (Week 12) (Week 12) (p 0.01); BARI 4 mg vs. BARI 4 BARI vs. PBO: 70% vs. 40% (p < 0.001); (Week 12) BARI 2 mg vs. PBO: 49% vs. 27% (p < 0.001); ≤ Main results mg + MTX: 77% vs. 78% BARI vs. ADA: 70% vs. 61% (p = 0.014) BARI 2 mg vs. PBO: 66% vs. 39% BARI 4 mg vs. PBO: 55% vs. 27% (p < 0.001) Currently recruiting (ACR20): (Week 52) (week 24) (p 0.001); BARI 4 mg vs. PBO: (Week 24) ≤ BARI 4 mg vs. MTX: 73% vs. 56% BARI vs. PBO: 74% vs. 37% (p < 0.001); 62% vs. 39% (p 0.001) BARI 2 mg vs. PBO: 45% vs. 27% (p 0.001); ≤ ≤ (p 0.05); BARI 4 mg vs. BARI 4 BARI vs. ADA: 74% vs. 66% (p 0.05) BARI 4 mg vs. PBO: 46% vs. 27% (p 0.001) ≤ ≤ ≤ mg + MTX: 73% vs. 73% The table summarizes the design and outcomes of baricitinib phase III confirmatory studies for rheumatoid arthritis patients (RA: rheumatoid arthritis; ACR: American College of Rheumatology; ACR20: improvement by 20% from baseline of core set parameters; ADA: adalimumab; BARI: baricitinib; cDMARD: conventional disease-modifying anti-rheumatic drug; HAQ-DI: health assessment questionnaire-disability index; MTX: methotrexate; OLE: open label extension; mTSS: modified total Sharp score; PBO: placebo; SDAI: Ssimplified disease activity index; TNFi: tumor necrosis factor inhibitor; DAS28-CRP: disease activity score on 28 joints by C-reactive protein; sc q2w: subcutaneous injection every other week; sid: once a day). Biomolecules 2020, 10, 1002 12 of 40

4.3. Selected Populations

4.3.1. Pediatric Patients No published data are currently available concerning the use of baricitinib in the pediatric population affected by juvenile idiopathic arthritis (JIA), although three phase III clinical trials (ClinicalTrials.gov ID: NCT03773978, NCT03773965 and NCT04088396) are at present evaluating the efficacy and safety of baricitinib in patients aged from one or two years to less than 18 years and affected by polyarticular or systemic JIA. The achievement of the clinical endpoints in JIA is challenged by the disease’s unpredictable expression and course. JIA encloses polyhedral manifestations, which may variously involve joints (poly- and pauci-articular forms) or extra-articular sites, or have a systemic development with distinct autoantibody patterns [115]. Around two thirds of pediatric patients affected by JIA may progress to other forms of arthritis in adulthood, which include RA but also seronegative arthritis. However, pathogenic pathways in JIA and adult RA are largely overlapping [115], and the use of oral JAKi could be of great interest in this subset of patients, whose compliance to parenterally administrated drugs, like biologics, is often limited.

4.3.2. Selected Ethnic Groups Due to the differences between Asian and non-Asian populations in terms of genetic background [116], RA prevalence [117], demographic characteristics and clinical practice (patients with RA in Japan are often prescribed with lower doses of MTX compared with patients in the United States, US [118]), additional subgroup analyses of the four main phase III trials were performed in order to evaluate the efficacy and safety profile of baricitinib in 394 Japanese patients and to assess whether results in this ethnic cohort were consistent with those emerged in overall study population [119]. In all phase III RCTs, the safety and tolerability profile of baricitinib in Japanese patients appeared acceptable and generally consistent with results from the prior phase IIb study of baricitinib in Japan [120] and its long-term extension [121], and with the overall study population data. An increased tendency to develop HZV reactivation under baricitinib was however reported in this ethnic group [122].

4.4. Safety The safety profile of baricitinib, emerging from the registration clinical trials and further analyses [105,120,123–128] on more than 3400 RA patients receiving a single dose of the drug, showed an incidence rate of serious adverse events (SAE) of eight in 100 patient-years of exposure (PYE) and a mortality rate of 0.33/100 PYE. The majority of the reported adverse reactions were infections and observed in 1/10 cases. The most common infections (with a prevalence rate between 1/10 and ≥ 1/100) were , HZV reactivation, , urinary tract infections, and . Hypercholesterolemia was a dose-dependent event. However, following an increase in the first 12 weeks of treatment, LDL and HDL serum levels were reported to stabilize [128]. No differences in terms of major cardiovascular events (MACE) were recorded between baricitinib and placebo in RCTs [129]. Generally, after 16 weeks of treatment, an increase in alanine transaminase (ALT) and aspartate transaminase (AST) was observed, especially when MTX was administered in combination [77,85,104]. RCTs also evidenced an alteration in the blood count of platelets and blood value. In particular, platelet counts may increase in the first two weeks of therapy and then stabilize. Hemoglobin may initially decrease and then slowly increase [77,104]. Japanese patients were reported to have a higher risk of HZV infection compared to general population [122]. Evidence on long-term safety still relies on the completion of the ongoing long-term extension studies, as well as pharmacovigilance real-life data. The most common adverse drug reactions during baricitinib therapy reported by Medical Dictionary for Regulatory Activities (MedDRA) system organ Biomolecules 2020, 10, 1002 13 of 40 class in Eudravigilance, Food and Drug Administration (FDA) adverse event reporting system (FAERS), and Vigiacess database, updated until 28 February 2020, are presented in Table3.

Table 3. List of adverse reactions reported under treatment with baricitinib by MedDRA system organ class in Eudravigilance, FAERS and Vigiacess database.

Number of Individual Cases by Reaction Groups (Updated on 28 February 2020) Reaction Groups VigiAccess Eudravigilance FAERS Blood and disorders 176 (2.0%) 98 (3.5%) 46 (2.2%) Cardiac disorders 117 (1.3%) 57 (2.1%) 54 (2.6%) Congenital, familial and genetic disorders 1 (0.0%) 1(0.0%) 2 (0.1%) Ear and labyrinth disorders 41 (0.5%) 15 (0.5%) 11 (0.5%) Endocrine disorders 7 (0.1%) 1 (0.0%) 1 (0.0%) Eye disorders 110 (1.3%) 36 (1.3%) 32 (1.6%) Gastrointestinal disorders 879 (10.0%) 310 (11.2%) 181(8.8%) General disorders and administration site conditions 1308 (14.9%) 326 (11.8%) 282(13.7%) Hepatobiliary disorders 66 (0.8%) 42 (1.5%) 46 (2.2%) Immune system disorders 88 (1.0%) 15 (0.5%) 26 (1.3%) Infections and infestations 2095 (23.9%) 615 (22.2%) 371 (18.0%) Injury, poisoning and procedural complications 348 (4.0%) 77 (2.8%) 91(4.4%) Metabolism and nutrition disorders 162 (1.8%) 75 (2.7%) 28 (1.4%) Musculoskeletal and connective tissue disorders 889 (10%) 151 (5.4%) 129 (6.3%) Neoplasms benign, malignant and unspecified (including cyst 99 (1.1%) 70 (2.5%) 100 (4.9%) and polyps) Nervous system disorders 566 (6.5%) 175 (6.3%) 158 (7.7%) Pregnancy, puerperium and perinatal conditions 4 (0.0%) 4 (0.1%) 1 (0.0%) Product issues 3 (0.0%) 0 (0.0%) 3 (0.1%) Psychiatric disorders 206 (2.3%) 56 (2.0%) 47 (2.3%) Renal and urinary disorders 134 (1.5%) 40 (1.4%) 37 (1.8%) Reproductive system and breast disorders 49 (0.6%) 16 (0.6%) 5 (0.2%) Respiratory, thoracic and mediastinal disorders 612 (7%) 247 (8.9%) 170 (8.3%) Skin and subcutaneous tissue disorders 504 (5.7%) 199 (7.2%) 89 (4.3%) Surgical and medical procedures 60 (0.7%) 1 (0.0%) 73 (3.5%) Vascular disorders 248 (2.8%) 147 (5.3%) 75 (3.6%) Total 8772 (100%) 2774 (100%) 2058 (100%)

4.5. Pharmacoeconomics According to two pharmacoeconomic studies, baricitinib has been considered a cost-effective treatment for RA patients with a previous inadequate response or intolerance to cDMARD therapy compared to adalimumab [130,131]. The same result was confirmed in the analysis including the hypothetical discount scenario of market entry of adalimumab [130]. From a US budget analysis, baricitinib was considered an equally effective and less expensive option compared to other biologic (b)DMARDs in RA patients with an active disease and an inadequate response to previous anti-TNF agents [132]. Although international recommendations did not express a definite ranking, a single technology appraisal from the National Institute for Health and Care Excellence (NICE) estimated an incremental cost-effectiveness ratio (ICER) for baricitinib, in combination with MTX, vs. intensive cDMARDs being £37,420 per quality-adjusted life year gained (QALY). This value is included in the range usually considered by NICE as a cost-effective use of national health service resources. The use of baricitinib in combination with MTX was judged less cost-effective than RTX plus MTX by NICE. Consequently, it was recommended as an option for patients with severe RA who can tolerate MTX if: (1) they have cDMARD inadequate response; (2) they have an anti-TNF inadequate response and rituximab (RTX) in combination with MTX is not an option; or (3) they have an anti-TNF inadequate response and have already been treated with RTX. NICE also recommended baricitinib in monotherapy or in combination with MTX as a cost-effective use of National Health Service resources in patients with severe RA, except for patients with inadequate response to anti-TNF who are RTX-eligible. In the latter case, the ICER for Biomolecules 2020, 10, 1002 14 of 40 biosimilars, and adalimumab, all in combination with MTX, were lower than £30,000 per QALY compared with baricitinib in combination with MTX [133].

5. Tofacitinib

5.1. Chemical Structure, Pharmacokinetics, Pharmacodynamics and Mechanism of Action Tofacitinib (IUPAC name: 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino] piperidin-1-yl]-3-oxopropanenitrile) is a pyrrolopyrimidine, a N-acylpiperidine, a nitrile and a tertiary amino compound. As citrate salt, it is soluble in dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL, slightly soluble in water at a concentration of 2.9 mg/mL after warming, and very slightly soluble in 99.5% ethanol [134]. Tofacitinib is a non-selective first generation JAKi. The drug has three labeled indications: RA, (PsA) and ulcerative colitis (UC), and acts by inhibiting JAK1, JAK2, JAK3 and, to a lesser extent, TYK2. Tofacitinib is orally administered at a dosage of 5 mg twice a day. After oral administration, it is rapidly absorbed with a Tmax of 0.5–1.0 h, and has a half-life of 3.2 h [135]. The drug has a dose-proportional pharmacokinetics. The steady-state is obtained 24–48 h after the first dose. Food decreases Cmax by 32% without affecting the area under the curve (AUC). The mean volume of distribution is 87 L after i.v. administration, with an equal distribution in red blood cells and plasma. About 40% of the drug is bound to plasma proteins. Tofacitinib inhibition of STAT is reversible 24 h after the cessation of treatment, while it lasts for 2 weeks in patients who received the treatment for a minimum of 4 consecutive weeks [82]. In ex-vivo experiments, it has been shown that tofacitinib decreases the expression of the IL-6 gene after a treatment period of 12 to 24 weeks, having instead a variable effect on that of IL-8, TNF-α and IL-10 genes [82]. In a study on RA patients, the serum levels of TNF-α, IL-17, IL-6, and IFN-γ significantly decreased following a four-week treatment with tofacitinib, whilst those of IL-35, mirroring Treg response, augmented [136]. Similar results were reported in responder psoriatic patients after 4 weeks of treatment [137]. In addition, in vitro studies exposing synovial membrane samples of PsA patients to the drug, further demonstrated an additional anti-angiogenic and anti-migrational effect [138,139]. Of note, tofacitinib suppresses in vitro the action of antigen presenting cells, by reducing the expression of the costimulatory molecules CD80/CD86 and by preventing the release of type I interferon [140]. Treatment with tofacitinib up to six months has been associated with a dose-dependent effect on white blood cells of RA individuals, including a reduction in natural killer (NK) cells (usually at 8–10 weeks of therapy and with a spontaneous reconstitution within 2–6 weeks after the discontinuation of treatment) and an increase in B cell count [141]. In healthy volunteers, no significant change in T-lymphocyte and their subsets has been reported in the short-medium term, whereas a prolonged treatment (approximately 5 years) has been associated with a reduction in T cells and an increase in NK cells from baseline [142]. Of note, lymphocyte subsets normalize after the temporary discontinuation of treatment and have not been associated with serious or opportunistic infections or with HZV reactivation. Although influencing the count of B cells in RA patients, tofacitinib seems not to affect the production of antibodies in healthy individuals [142]. However, a study conducted on umbilical cord blood B cells and B lymphocytes of tofacitinib-treated patients evidenced that the drug may interfere with the maturation of B naive lymphocytes [143]. Tofacitinib is the only non-selective JAKi able to prevent the tolerogenic IL-27 pathway, in turn mediated by TYK2 signaling [144]. However, the drug is also able to hamper the STAT3-mediated differentiation of Th17 lymphocytes, thus counteracting the development of autoreactive cells [145]. As for baricitinib, tofacitinib has a hepatic metabolism through the cytochrome CYP3A4 [135], and, to a lesser extent, CYP2C19. Accordingly, also in this case, the co-administration of tofacitinib with CYP3A4 and CYP2C9 inhibitors or inducers should be carefully monitored. Biomolecules 2020, 10, 1002 15 of 40

Contrary to baricitinib, the of tofacitinib is mainly via the gastro-intestinal apparatus with only 30% of inactive metabolites excreted in urine. The renal and hepatic clearance are 124 mL/min and 289 mL/min, respectively [134,135]. Therefore, the use of tofacitinib appears safer than that of baricitinib in patients with an impairment of the renal function. Tofacitinib can be also used in patients with a moderate hepatic failure, though a dose reduction is required [146].

5.2. Efficacy The clinical efficacy of oral tofacitinib 5 mg and 10 mg twice daily as monotherapy or in combination with cDMARDs for the treatment of RA has been reported in six pivotal randomized, double-blind, multicentric phase III clinical studies (ORAL Solo—A3921045 [147]; ORAL Start—A3921069 [148]; ORAL Sync—A3921046 [149]; ORAL Scan—A3921044 [150]; ORAL Standard—A3921064 [151]; ORAL Step—A3921032 [152]), and in two open-label long-term extension studies (ORAL Sequel Study—A3921024 [87] and the Japanese study A3921041 [153]). Two studies (ORAL Scan and ORAL Start) assessed radiographic outcomes [148,150]. Recruited patients included MTX-naïve subjects (ORAL Start trial), inadequate responders to MTX or other cDMARDs (ORAL Scan, ORAL Sync and Oral Standard) and inadequate responders to biologics (ORAL Step, ORAL Sync and ORAL Solo). Globally, the studies confirmed the clinical and radiographic efficacy of tofacitinib at both the doses of 5 and 10 mg twice daily. In the ORAL Start trial [148], the coprimary efficacy endpoints, consisting of the mean change from baseline of the modified total Sharp score (mTSS) and the ACR70 response rate at month 6, were met: of note, the mean change in the mTSS from baseline was significantly smaller in the tofacitinib groups than in the MTX group, although changes were modest in all the three groups. Significant clinical and radiographic improvements were reported as early as month 1 and sustained over 24 months. Similarly, in the ORAL Solo, ORAL Step, ORAL Standard, and ORAL Scan trials, benefits concerning DAS28-erythrocyte sedimentation rate (ESR) scores, ACR20-ACR50-ACR70 response rates, HAQ-DI and PROs scores were already reported at month 3 [147,150–152]. The ORAL Standard trial, conducted on RA patients who were MTX-non responder, compared tofacitinib to adalimumab [151]. Although a formal non-inferiority comparison among the active treatments was not made, the clinical efficacy of tofacitinib resulted numerically similar to that observed with adalimumab, with clinical responses achieved in both the two treatment arms by 1 month and sustained to month 12. These results are in contrast to what was reported with baricitinib in the RA-BEAM trial, in which baricitinib showed superiority over adalimumab in the ACR20 response rate and mean change in DAS28-CRP at week 12 [119]. Although studies differed in the design, the apparent higher efficacy of baricitinib could be reconducted to its mechanism of action. Being more selective on JAK1 than tofacitinib, baricitinib strongly prevents the secretion of IL-6, and the following IL-6 blockade may play a more powerful therapeutic effect than the inhibition of TNF-α [154]. Anyway, to date, no published data on the direct comparison between baricitinib and tofacitinib are available, although a trial (ClinicalTrials.gov ID NCT03755466) is ongoing. The phase 3b/4 ORAL Strategy trial [155], conducted in RA patients with active disease despite a previous treatment with MTX, showed comparable results in terms of ACR responses, DAS28-ESR remission and low disease activity rates at month 6 between the tofacitinib (5 mg twice a day) and the adalimumab arm. Of note, in this study, tofacitinib monotherapy showed less efficacy than in combination with MTX. Physical function (HAQ-DI score and other PROs) improved from baseline to a generally similar extent in patients receiving tofacitinib plus MTX, adalimumab plus MTX or tofacitinib monotherapy [156]. The long-term effect of tofacitinib has been explored in the ORAL Sequel study, including 4481 patients who had previously completed a phase I, II, or III study of tofacitinib and received open-label tofacitinib 5 mg or 10 mg twice a day [87]. Statistical analysis demonstrated that both clinical and functional index scores were maintained over the time between months 1 and 96 and were Biomolecules 2020, 10, 1002 16 of 40 generally similar with tofacitinib 5 mg and 10 mg twice a day. Also, CDAI- and SDAI-defined remission were still reported in approximately one third of patients at month 96, with a limited structural damage progression during longer-term therapy [157]. A synthesis of the main characteristics of clinical trials on tofacitinib is reported in Table4.

Table 4. Tofacitinib phase III clinical trials in moderate to severe rheumatoid arthritis.

ORAL Start ORAL Solo ORAL Sync ORAL Scan ORAL Standard ORAL Strategy ORAL Step Trial MTX-naïve c/bDMARD-IR c/bDMARD-IR MTX-IR MTX-IR MTX-IR TNF-IR (n = 958) (n = 611) (n = 795) (n = 797) (n = 717) (n = 1146) (n = 399) Active RA Moderate to patients with severe RA Active RA Active RA inadequate Active RA Active RA patients MTX-naïve patients with patients response to patients patients with Participants patients with ≥ inadequate receiving 1 c/bDMARD receiving stable receiving stable inadequate active RA response to 1 background receiving ≥ doses of MTX doses of MTX response to c/bDMARD MTX stable doses of anti-TNF antimalarial drugs Type of Combination Combination Combination Combination Monotherapy Monotherapy Monotherapy therapy therapy therapy therapy therapy Active ADA + MTX MTX /// ADA / Comparator non-inferiority Background None None cDMARD MTX MTX None MTX treatment (1) TOFA 5 mg (1) TOFA 5 mg (1) TOFA 5 mg (1) TOFA 5 mg bid (1) TOFA bid bid bid (2) TOFA 10 mg 5 mg bid (2) TOFA 10 mg (2) TOFA 10 mg (2) TOFA 10 bid (2) TOFA (1) TOFA bid bid (1) TOFA 5 mg mg bid (3) PBO 10 mg bid 5 mg bid (3) PBO (3) PBO bid (3) PBO advanced to (3) PBO Arms (2) TOFA advanced to advanced to (2) TOFA 10 mg advanced at TOFA 5 mg bid advanced to 10 mg bid TOFA 5 mg bid TOFA 5 mg bid bid 3 months to or 10 mg bid at TOFA 5 mg (3) MTX or 10 mg bid at or 10 mg bid at (3) ADA + MTX TOFA 5 mg 6 months bid or 10 mg 6 months 6 months bid or 10 mg (3 months for bid at (3 months for (3 months for bid non-responders) 3 months non-responders) non-responders) (4) ADA Duration 24 6 12 24 12 12 6 (months) Features X-Rays X-Rays ACR20 ACR20 ACR20 ACR20 ∆mTSS ACR20 DAS28-ESR < DAS28-(ESR) < ∆mTSS HAQ-DI DAS28-ESR < HAQ-DI Coprimary 2.6 2.6 ACR50 ACR70 DAS28-ESR < 2.6 DAS28-ESR endpoints (month 6) (month 6) (month 6) (month 6) 2.6 (month 6) < 2.6 HAQ-DI HAQ-DI (month 3) HAQ-DI (month 3) (month 3) (month 3) (month 3) Biomolecules 2020, 10, 1002 17 of 40

Table 4. Cont.

ORAL Start ORAL Solo ORAL Sync ORAL Scan ORAL Standard ORAL Strategy ORAL Step Trial MTX-naïve c/bDMARD-IR c/bDMARD-IR MTX-IR (n = MTX-IR (n = MTX-IR (n = TNF-IR (n = (n = 958) (n = 611) (n = 795) 797) 717) 1146) 399) (Month 6) ACR20 (% pts): 71.3 (Month 6) (p < 0.001) (Month 6) ACR20 (%pts): (Month 3) TOFA 5 mg; ACR20 (%pts): 51.5 (p < 0.001) (Month 6) ACR20 (% 76.1 (p 0.01) ≤ 52.7 (p < 0.001) TOFA 5 mg + ACR20 (%pts): pts): 41.7 TOFA 10 mg; TOFA 5 mg+ MTX; 51.5 (p < 0.001) (p 0.01)) 50.5 MTX. ≤ cDMARD; 56.6 61.8 (p < 0.001) TOFA 5 mg + (Month 6) TOFA 5 mg ACR70 (% (Month 3) (p < 0.001) TOFA 10 mg + MTX; 52.6 ACR20 (% pts): + MTX; 48.1 pts): 25.5 ACR20 (% pts): TOFA 10 mg + MTX; 25.3 PBO (p < 0.001) TOFA 65 TOFA 5 mg; (p < 0.001) (p < 0.001) 59.8 (p < 0.001) cDMARD; 31.2 + MTX. 10 mg + MTX; 73.1 TOFA 5 mg TOFA 10 mg TOFA 5 mg; TOFA 5 mg; PBO + DAS28-ESR < 47.2 (p < 0.001) + MTX; 71 + MTX; 24.4 37.7 (p 0.01) ≤ 65.7 (p < 0.001) cDMARD. 2.6 (% pts): 7.2 ADA + MTX; ADA+ MTX. PBO. + MTX TOFA 10 mg; TOFA 10 mg; HAQ-DI TOFA 5 mg + 28.3 PBO+ MTX. ACR50 (% pts): HAQ-DI 12 MTX. 26.7 PBO. (change from MTX; 16.0 HAQ-DI (change 38.3 TOFA 5 mg; (change HAQ-DI HAQ-DI BL): 0.44 (p < 0.001) from BL): 46 TOFA 5 mg + from BL): (change from − (change from (p < 0.001) TOFA 10 mg + 0.55 (p < 0.001) MTX; 44 ADA+ 0.43 BL): 0.8 − − − BL): 0.5 TOFA 5 mg + MTX; TOFA 5 mg+ MTX. (p < 0.001) (p < 0.001) − (p < 0.001) cDMARD; 1.6 PBO + MTX. MTX; 0.61 HAQ-DI TOFA 5 mg + Main results TOFA 5 mg; − TOFA 5 mg; 0.53 HAQ-DI (p < 0.001) TOFA (change from MTX; 0.46 0.9 − − − 0.57 (p < 0.001) (change from 10 mg + MTX; BL): 0.54 TOFA (p < 0.001) (p < 0.001) − − (p < 0.001) TOFA 10 mg+ BL): 0.49 (p < 0.001) 5 mg; 0.59 TOFA 10 mg TOFA 10 mg; − − TOFA 10 mg; cDMARD; 0.40 TOFA ADA + MTX; TOFA 5 mg + + MTX; 0.6 MTX. − − 0.19 PBO. 0.21 PBO + 5 mg + MTX; 0.24 PBO + MTX; 0.54 0.18 PBO + DAS28-ESR < − − − − − DAS28-ESR < cDMARD. 0.54 (p < 0.001) MTX. ADA + MTX. MTX. 2.6 (% pts): − 2.6 DAS28-ESR < TOFA 10 mg + DAS28-ESR < 2.6 DAS28-ESR < DAS28-ESR 14.6 (p 0.05) ≤ (% pts): 5.6 2.6 (% pts): 8.5 MTX; (% pts): 7 2.6 (% pts): 10.4 < 2.6 TOFA 5 mg; TOFA 5 mg; (p 0.01) TOFA 0.15 PBO + (p 0.05) TOFA 5 TOFA 5 mg; 12 (% pts): 6.7 21.8 (p 0.01) ≤ − ≤ ≤ 8.7 TOFA 5 mg + MTX. mg + MTX; 12.5 TOFA 5 mg + (p 0.05) TOFA 10 mg; ≤ 10 mg; 4.4 PBO cDMARD; 12.5 ∆mTSS (from (p < 0.001) TOFA MTX; 12.4 ADA TOFA 5 mg 7.6 MTX. (p < 0.001) baseline): 0.12 10 mg + MTX; 6.7 + MTX. + MTX; 8.8 ∆mTSS (from TOFA 10 mg + TOFA 5 mg + (p 0.05) ADA + (p 0.05) baseline): 0.2 ≤ ≤ cDMARD; 2.7 MTX; 0.06 MTX; 1.1 PBO + TOFA 10 mg (p < 0.001) PBO + (p 0.05) TOFA MTX + MTX; 1.7 TOFA 5 mg; ≤ cDMARD 10 mg + MTX; PBO + MTX <0.1 0.47 PBO + MTX (p < 0.001) TOFA 10 mg; 0.8 MTX The table summarizes the design and outcomes of tofacitinib phase III confirmatory studies for rheumatoid arthritis patients (RA, rheumatoid arthritis; ACR: American College of Rheumatology; ACR20: improvement by 20% from baseline of core set parameters; ADA: adalimumab; cDMARD: conventional disease-modifying anti-rheumatic drug; HAQ-DI: health assessment questionnaire-dDisability index; MTX: methotrexate; OLE: open label extension; mTSS: modified total Sharp score; PBO: placebo; SDAI: Simplified Disease Activity Index; TOFA: tofacitinib; DAS28-ESR: disease activity score on 28 joints by erythrocyte sedimentation rate; BL: baseline; bid: twice a day).

5.3. Selected Populations

5.3.1. Pediatric Patients Evidence on the use of tofacitinib in pediatric population is limited. Few preliminary studies have been performed to establish the safety and pharmacokinetics of tofacitinib in patients affected by JIA [158]. The profile of efficacy and safety of tofacitinib 1–5 mg twice a day has been investigated in a recently completed phase III, randomized withdrawal, double-blind, placebo-controlled study in JIA patients (2 to <18 years) (A3921104; ClinicalTrials.gov ID NCT02592434; data not published). A phase II-III, long-term, open-label, follow-up study is also ongoing for those JIA patients who have previously participated in qualifying/index JIA studies of tofacitinib, including phase I studies (A3921165; Clinicaltrials.gov: NCT01500551), while a phase III, randomized, withdrawal, double blind, placebo-controlled study is currently recruiting patients with systemic JIA (A3921165; ClinicalTrials.gov ID NCT03000439). Recently, Huang et al. [159] reported the case of a 13-year-old girl with recalcitrant systemic JIA non responder to glucocorticoids, cDMARDs and etanercept, who was prescribed with tofacitinib 2.5 mg twice daily. The authors observed a stable improvement of both articular and systemic symptoms after Biomolecules 2020, 10, 1002 18 of 40

2 months of treatment, with the achievement of a complete remission at month 3. Interestingly, no disease relapse or safety concerns occurred throughout the six months of follow-up.

5.3.2. Selected Ethnic Groups The efficacy and safety profile of tofacitinib has been investigated in the long-term extension study A3921041 (ClinicalTrial.gov ID: NCT00661661), conducted in 486 Japanese patients who had participated to prior phase II or phase III studies of tofacitinib as monotherapy or in combination with MTX. Final results demonstrated a sustained efficacy profile of tofacitinib (with or without MTX), consistent with that observed in the main phase III studies, along with a stable safety profile, although a higher risk of HZV reactivation has been highlighted in Japanese patients compared to the general population [153].

5.4. Indirect Studies Comparing Tofacitinib Efficacy As no robust evidence is available concerning direct comparisons between biologics in RA clinical trials, indirect comparisons (network meta-analysis and registries) could provide the most relevant and comprehensive data for a relative efficacy assessment. One systematic review and network meta-analysis assessed the safety and effectiveness of biologics (, adalimumab, , certolizumab pegol, etanercept, , infliximab, rituximab and ) and tofacitinib in RA patients who had an inadequate response to cDMARD treatment [160]. Data, collected from 79 trials and including 32,874 participants, failed to show a superiority of mono- or combo-therapy with tofacitinib in terms of improvement in the ACR50 response compared to biologic agents. Similarly, in patients taking cDMARDs, there was no significant difference between the likelihood of having better HAQ-DI scores following the administration of biologics or tofacitinib. Another systematic review and network meta-analysis, assessing the safety and efficacy of tofacitinib in biologic-resistant patients, included data from 12 trials extrapolated on a cohort of 3364 participants [161]. Data analysis showed that for every 100 patients treated with tofacitinib plus MTX instead than with MTX alone, 19 additional patients would experience significant improvement in their RA symptoms (based on ACR50 response) compared to 16 patients treated with a biological agent plus MTX. Similarly, it was estimated that for every 100 patients treated with tofacitinib plus MTX instead than MTX alone, 6 extra patients would achieve DAS44 or DAS28 remission compared to 10 extra patients treated with a biologic drug plus MTX. A network meta-analysis by Vieira et al., analyzing 5 trials for a total of 2136 patients, revealed that tofacitinib at a dose of 5 mg twice a day combined with MTX was similar to biologics (abatacept, golimumab, rituximab, and tocilizumab) combined with cDMARDs in terms of the relative risk (RR) of ACR20, ACR50, and ACR70 responses and change from baseline in HAQ-DI scores [162]. These findings were confirmed by another network meta-analysis, which aimed to assess the efficacy of tofacitinib at a dose of 5 and 10 mg twice a day given either as monotherapy or combined with MTX or other cDMARDs, in comparison with biologic and synthetic therapies (abatacept, adalimumab, anakinra, certolizumab pegol, etanercept, golimumab, infliximab, tocilizumab, and baricitinib) at 24 weeks [163]. Tofacitinib, given twice a day at a dose of 5 mg, showed comparable results to those observed with the use of other biologic monotherapies (tocilizumab, certolizumab, etanercept and adalimumab) in terms of ACR20 and ACR70 response rate at week 24. In addition, the combo-therapy of tofacitinib 5 and 10 mg twice a day with MTX or other cDMARDs was more effective than certolizumab 400 mg every 4 weeks plus MTX or other cDMARDs in the achievement of the ACR70 response. Tofacitinib 10 mg twice a day revealed higher efficacy in the ACR20 response rate than etanercept, abatacept, and infliximab (all combined with cDMARDs). At 24 weeks, the ACR50 response rate indicated significantly higher efficacy of tofacitinib 5 and 10 mg twice a day compared to baricitinib (all administered with concomitant therapies). Also, a higher percentage of patients under tofacitinib Biomolecules 2020, 10, 1002 19 of 40 reached the ACR70 response at 24 weeks compared to adalimumab, abatacept, etanercept, infliximab and baricitinib 2 mg daily plus cDMARDs. Recently, results from the Swiss RA registry on a cohort of 2600 patients, of whom 806 treated with tofacitinib, were published [164]. The drug retention rate of tofacitinib was higher than that of anti-TNF agents but comparable with that of non-anti-TNF biologics (abatacept or anti-IL-6 agents). The use of cDMARDs improved the effectiveness of anti-TNF drugs, but not that of tofacitinib or non-anti-TNF biologics, supporting the efficacy of these drugs in monotherapy. Another recent study extrapolated real-life data from the Corrona US registry in order to evaluate the effectiveness of tofacitinib vs. anti-TNF drugs in RA patients [165]. A total of 558 subjects treated with tofacitinib from 2012 to 2016 were included. Interestingly, tofacitinib, either as mono- or combo-therapy, proved to be still effective even in patients with long-standing RA and receiving the drug as third or fourth option.

5.5. Safety A safety report from phase II and III RTCs of tofacitinib showed that the majority of adverse events had a mild to moderate severity [147–152,166]. The most common side effects were nasopharyngitis (prevalence 1/10), lower respiratory tract infections, HZV reactivation, urinary tract infections, ≥ , increase in creatinine serum levels and enzymes, dyslipidemia with a modest and reversible increase of LDL and HDL levels [167], neutropenia, anemia, oedema, , and dyspnea (prevalence between 1/10 and 1/100) [134]. The decrease in neutrophil count has been considered to be dose-dependent [168], and, when it occurs in a moderate manner, it may be associated with a better clinical response in RA [82]. Generally, neutrophil count is stabilized after three months since the start of a treatment with tofacitinib. Hemoglobin value has been reported to initially decrease and then to slowly increase during the treatment [87,169]. As for baricitinib, the concomitant administration of MTX may increase the risk of hyper-transaminasemia, and Japanese and Korean patients were reported to have a higher incidence of HZV infection than other populations [153]. Among SAE, the most common (prevalence between 1/10 and 1/100) were infections and malignancies (lympho-proliferative disorders and non-melanoma skin ) [170,171]. An integrated analysis of pooled safety data obtained from phase II and III RCTs on 5671 treated patients reported 107 malignancies developing under tofacitinib treatment [172]. Malignancies mainly affected the lungs, breast, or lymphoid organs, however they were stable over time and demonstrated an incidence in line with that reported in RA patients with a moderate to severe disease activity. Of note, in May 2019 the European Medicines Agency (EMA)’s Safety Committee (PRAC) put a warning on the use of tofacitinib 10 mg twice a day in individuals at high risk of lung thromboembolic events. These include patients suffering from heart failure, cancer, inherited blood clotting disorders or a history of blood clots, or subjects taking combined hormonal contraceptives, hormone replacement therapy or who undergo major surgery. This warning derived from the ongoing phase IV study A3921133 (ClinicalTrials.gov ID: NCT02092467), preliminarily reporting an increased risk of and in RA patients assuming tofacitinib 10 mg twice a day. Although this dosage is currently not recommended in RA patients in clinical practice, patients at risk of thrombotic events should be carefully monitored [173]. The most common adverse drug reactions reported by MedDRA system organ class in Eudravigilance, FAERS, and VigiAccess database are reported in Table5. Biomolecules 2020, 10, 1002 20 of 40

Table 5. List of adverse reactions reported under treatment with tofacitinib by MedDRA system organ class in Eudravigilance, FAERS and Vigiacess database.

Number of Individual Cases by Reaction Groups (Updated on 28 February 2020) Reaction Groups VigiAccess Eudravigilance FAERS Blood and lymphatic system disorders 959 (0.8%) 749 (1.3%) 1276 (1.0%) Cardiac disorders 1302(1.1%) 1240 (2.1%) 1870 (1.4%) Congenital, familial and genetic disorders 51(0.0%) 51 (0.1%) 91 (0.1%) Ear and labyrinth disorders 792 (0.7%) 442 (0.8%) 985 (0.7%) Endocrine disorders 157 (0.1%) 145 (0.2%) 262 (0.2%) Eye disorders 1681 (1.4%) 1173 (2.0%) 2176 (1.6%) Gastrointestinal disorders 10,951 (9.4%) 4894 (8.4%) 12,200 (9.1%) General disorders and administration site conditions 29,273 (25.2%) 10,533 (18.0%) 31,152 (23.2%) Hepatobiliary disorders 595 (0.5%) 537 (0.9%) 939 (0.7%) Immune system disorders 1809 (1.6%) 1052 (1.8%) 2560 (1.9%) Infections and infestations 16,042 (13.8%) 8940 (15.3%) 16,908 (12.6%) Injury, poisoning and procedural complications 8189 (7.0%) 5028 (8.6%) 10,407 (7.8%) Metabolism and nutrition disorders 1385 (1.2%) 980 (1.7%) 1728 (1.3%) Musculoskeletal and connective tissue disorders 12,602 (10.8%) 6497 (11.1%) 15271 (11.4%) Neoplasms benign, malignant and unspecified 1587 (1.4%) 1611 (2.8%) 2358 (1.8%) (including cyst and polyps) Nervous system disorders 8910 (7.7%) 4252 (7.3%) 9929 (7.4%) Pregnancy, puerperium and perinatal conditions 29 (0.0%) 33 (0.1%) 59 (0.0%) Product issues 86 (0.1%) 49 (0.1%) 112 (0.1%) Psychiatric disorders 3026 (2.6%) 1502 (2.6%) 3567 (2.7%) Renal and urinary disorders 1508 (1.3%) 1077 (1.8%) 1869 (1.4%) Reproductive system and breast disorders 405 (0.3%) 223 (0.4%) 460 (0.3%) Respiratory, thoracic and mediastinal disorders 6594 (5.7%) 3465 (5.9%) 7725 (5.8%) Skin and subcutaneous tissue disorders 5706 (4.9%) 2306 (3.9%) 6684 (5.0%) Surgical and medical procedures 865 (0.7%) 216 (0.4%) 1289 (1.0%) Vascular disorders 1832 (1.6%) 1516 (2.6%) 2403 (1.8%) Total 116,336 (100%) 58,511 (100%) 134,280 (100%)

5.6. Pharmacoeconomics A few economic evaluations were carried out in the US for tofacitinib, showing limited additional costs or even potential cost saving [174,175]. When given in monotherapy in MTX-intolerant patients, or with MTX in anti-TNF-intolerant patients, tofacitinib proved to be a less costly option compared to other bDMARDs as second-line treatment [176]. A treatment strategy with tofacitinib as either second- or third-line therapy after MTX may be a cheaper option, compared with the introduction of tofacitinib as fourth-line after cycling through 2 anti-TNF agents [177]. This finding was in line with a previous economic evaluation of tofacitinib vs. a set of biologic agents. This study showed that tofacitinib 5 mg twice a day was a cost-effective treatment option for RA compared to adalimumab or etanercept. This was due to lower costs per patient when the drug was given in monotherapy or in combination with other cDMARDs in MTX-intolerant patients. The same was observed in anti-TNF-intolerant patients, in whom tofacitinib plus MTX was more cost-effective than adalimumab plus MTX [178]. Such results were confirmed by Kulikov et al., who assessed that therapy with tofacitinib could reduce the annual cost of RA treatment from 8846€ to 2037€ per patient in comparison with other bDMARDs [176]. According to a NICE Appraisal, tofacitinib in combination with MTX is a cost-effective use of National Health Service resources in patients with severe RA with inadequate response to cDMARDs, except for etanercept biosimilar in combination with MTX [179]. In patients with severe RA with inadequate response to bDMARDs, tofacitinib in combination with MTX was more cost-effective only in the group of RTX non-eligible patients. Tofacitinib monotherapy showed a less expensive, though slightly less effective, profile than that of comparators, and its use may replace the combo-therapy with MTX in MTX-intolerant patients. In MTX-intolerant patients, tofacitinib and tocilizumab monotherapy extendedly dominated Biomolecules 2020, 10, 1002 21 of 40 a monotherapy with adalimumab and etanercept biosimilars [179]. Another recent study indicated tofacitinib as a dominant strategy (more effective and less costly) in patients affected by moderate to severe RA who are refractory to conventional and biologic drugs in second line and third line treatments, respectively [180], compared to other alternatives.

6. Second Generation JAKi Considering the FDA and EMA approval of baricitinib and tofacitinib for the treatment of adults affected by moderate to severe anti-TNF resistant RA and for the treatment of moderate to severe MTX-refractory RA, active PsA and moderate to severe anti-TNF-refractory ulcerative colitis respectively, other JAKi have been developed for RA, reaching, in two cases, the market. Among them, upadacitinib was licensed in US and Europe and peficitinib authorized in Japan for the treatment of adult patients with moderate to severe active RA and an inadequate response or intolerance to MTX, while filgotinib and decernotinib are still under clinical investigation for RA. Itacitinib, a JAKi licensed for different therapeutic indications, has also been tested in RA in a phase II RCT. Current evidence concerning these compounds is reported in the next subparagraphs. A synopsis of the chemical structure and the main pharmacological properties of the first and second generation JAKi aforementioned, are instead provided in Figure3 and Table6. Biomolecules 2019, 9, x FOR PEER REVIEW 20 of 38

Figure 3. Molecular structure of first and second generation JAKi compared. Figure 3. Molecular structure of first and second generation JAKi compared.

Table 6. Synopsis of the main pharmacological properties of first and second generation JAKi. (JAKi, Janus kinase inhibitors; JAK, Janus kinase; TYK2, tyrosin kinase 2; sid, single dose; bid, twice daily; PK: pharmacokinetics; PD: pharmacodynamics; FDA: Food and Drug Administration; EMA: European Medicines Agency; Tmax: time to peak; bid: twice a day; sid; once a day; OAT: ornithine aminotransferase; CYP: cytochrome p450; US, United States; Eu, Europe; AUC, area under the curve; IC50, half maximal inhibitory concentration).

Drug First generation JAKi Second Generation JAKi active Baricitinib Tofacitinib Upadacitinib Decernotinib principle brand name Olumiant® Xeljanz® Rinvoq™ Smyraf® / / INCB028050 ASP015K, other name CP-690,550 ABT-494 GLPG0634/GS-6034 VX-509 LY3009104 JNJ-54781532 JAK1 JAK1 JAK3 JAK3 target JAK1 JAK1 JAK3 JAK2 JAK2 JAK1 TYK2 150 mg sid or 100 mg depending on Dose 2 mg sid 5 mg bid 15 mg sid 100 mg or 200 mg sid 50–150 mg bid the patient’s condition 1 mg once daily in patients with No dose adjustment No dose adjustment in creatinine in patients with patients with mild (50–80 No dose clearance between mild, moderate or mL/min) or moderate (30–49 adjustment renal 30 and 60 mL/min severe renal mL/min) renal impairment required in / / insufficiency Not recommended impairment 5 mg once daily in patient patients with renal in patients with Not tested in with severe renal impairment creatinine subjects with end impairment (<30 mL/min) clearance < 30 stage renal disease mL/min No dose adjustment in No dose adjustment No dose patients with mild (Child in patients with mild adjustment in Pugh A) hepatic 50 mg sid in (Child Pugh A) or patients with mild impairment patients with moderate (Child or moderate 5 mg once daily moderate liver Pugh B) hepatic hepatic recommended in patient dysfunction impairment / / impairment Not with moderate hepatic Contraindicated in Not recommended recommended in function (

Table 6. Synopsis of the main pharmacological properties of first and second generation JAKi. (JAKi, Janus kinase inhibitors; JAK, Janus kinase; TYK2, tyrosin kinase 2; sid, single dose; bid, twice daily; PK: pharmacokinetics; PD: pharmacodynamics; FDA: Food and Drug Administration; EMA: European Medicines Agency; Tmax: time to peak; bid: twice a day; sid; once a day; OAT: ornithine aminotransferase; CYP: cytochrome p450; US, United States; Eu, Europe; AUC, area under the curve; IC50, half maximal inhibitory concentration).

Drug First generation JAKi Second Generation JAKi active principle Baricitinib Tofacitinib Upadacitinib Peficitinib Filgotinib Decernotinib brand name Olumiant® Xeljanz® Rinvoq™ Smyraf® // INCB028050 other name CP-690,550 ABT-494 ASP015K, JNJ-54781532 GLPG0634/GS-6034 VX-509 LY3009104 JAK1 JAK1 JAK3 JAK3 target JAK1 JAK1 JAK3 JAK2 JAK2 JAK1 TYK2 150 mg sid or 100 mg Dose 2 mg sid 5 mg bid 15 mg sid depending on the patient’s 100 mg or 200 mg sid 50–150 mg bid condition No dose adjustment in patients 1 mg once daily in patients with mild (50–80 mL/min) or No dose adjustment in with creatinine clearance moderate (30–49 mL/min) renal patients with mild, moderate No dose adjustment required between 30 and 60 mL/min renal insufficiency impairment or severe renal impairment in patients with renal // Not recommended in 5 mg once daily in patient with Not tested in subjects with impairment patients with creatinine severe renal impairment end stage renal disease clearance < 30 mL/min (<30 mL/min) No dose adjustment in patients No dose adjustment in with mild (Child Pugh A) hepatic No dose adjustment in patients with mild or impairment patients with mild (Child 50 mg sid in patients with moderate hepatic 5 mg once daily recommended in Pugh A) or moderate (Child moderate liver dysfunction liver failure impairment Not patient with moderate hepatic Pugh B) hepatic impairment // Contraindicated in patient recommended in patients function (

Table 6. Cont.

Drug First generation JAKi Second Generation JAKi Most frequent: upper and lower airway infections; influenza; herpes zoster virus infection; urinary tract infections; abdominal pain; vomiting; Most frequent: upper Most frequent: throat and ; nausea; gastritis; rash; respiratory tract infections Most frequent: nose infections; herpes headache; anemia; leukopenia; (, sinus nasopharyngitis; virus infection; Most frequent: hyper-transaminasemia infections); nausea; cough; zoster virus infection; blood Most frequent: nausea; infections causing a sick nasopharyngitis; nausea; Uncommon: ; fever creatine kinase increase; headache; nasopharyngitis; stomach or diarrhea; bronchitis; headache; upper diverticulitis; pyelonephritis; Uncommon: serious lymphopenia diarrhea; upper respiratory urinary infections; respiratory tract infection cellulitis; infections; malignancies; Uncommon: pneumonia; tract infections; blood safety pneumonia; Uncommon: major adverse infection; viral gastroenteritis thrombosis; gastrointestinal ; epipharyngitis; cholesterol increase; thrombocytosis nausea; cardiac events pulmonary and other viral infections; blood perforations; altered upper respiratory tract alanine aminotransferase Uncommon: leukopenia; embolism; herpes zoster creatinine increase; blood laboratory parameters; infections bronchitis; increase Uncommon: increase in serum creatine virus infection; deep vein cholesterol increase; low density embryo-fetal toxicity influenza; cystitis serious infections kinase; high serum levels of thrombosis lipoprotein increase; weight Rare: major adverse cardiac Rare: ; gastrointestinal triglycerides; acne; weight increase events pulmonary embolism; perforation gain Rare: sepsis; disseminated venous thromboembolism tuberculosis involving bones and other organs; other unusual infections; joint infection Licensed therapeutic rheumatoid arthritis, psoriatic Under regulatory review rheumatoid arthritis rheumatoid arthritis rheumatoid arthritis indications arthritis, ulcerative colitis for rheumatoid arthritis Tmax 0,5–3 h; t1/2 3.82–10.9 h (after Tmax 1.1–2.1 h; t1/2 single/multiple twice daily 9.9–16.2 h PK Tmax 1.5 h; t1/2 12.5 h Tmax 0.5–1 h; t1/2 3.3 h Tmax 2–4 h; t1/2 8-14 h 25–450 mg dosages) N.A. (after multiple 30–200 mg Tmax 1–2; t1/2 (after twice daily dosages) single/multiple twice daily 30–300 mg dosages) IC50JAK1 5.9 nM; IC50JAK1 3.2 nM; IC50JAK1 3.9 nM; IC50JAK2 IC50JAK1 45 nM; IC50JAK2 IC50JAK1 10 nM; IC50JAK2 IC50JAK1 10 nM; IC50JAK2 IC50JAK2 5.7 nM; IC50JAK2 4.1 nM; 5.0 nM; IC50 109 nM; IC50JAK3 2.1 µM; 28 nM; IC50JAK3 810 nM; 10 nM; IC50JAK3 2.5 nM; IC50JAK3 420 nM; IC50JAK3 1.6 nM; IC50JAK3 0.71 nM; IC50Tyk2 IC50TYK2 4.7 µM IC50Tyk2 110 nM IC50TYK2 10 nM IC50TYK2 60 nM IC50TYK2 34 nM 4.8 nM CYP3A4 inhibitors (e.g., Verapamil (P-glycoprotein ketoconazole), medicinal inhibitor that increase AUC OAT3 inhibitors and products that result in both CYP3A4 inhibitors (e.g., and Cmax of peficitinib by CYP3A4 inhibitors (e.g., drug interactions moderate inhibition of CYP3A4 ketoconazole) and inducers 27–39%) / CYP3A4 inhibitors ketoconazole) and inducers as well as potent inhibition of (e.g., rifampicin) No clinically significant (e.g., rifampicin) CYP2C19 (e.g., fluconazole) and interaction with methotrexate CYP inducers and rosuvastatin /// Biomolecules 2020, 10, 1002 24 of 40

6.1. Upadacitinib Upadacitinib (IUPAC name: 3S,4R)-3-ethyl-4-(1,5,7,10-tetrazatricyclo[7.3.0.02,6]dodeca-2(6),3,7,9, 11-pentaen-12-yl)-N-(2,2,2-trifluoroethyl)pyrrolidine-1-carboxamide) was developed as a JAK1-selective inhibitor by exploiting differences in the non-conserved domains outside the active sites of JAK1 and JAK2. In cellular assays, upadacitinib displays 60 and 100 fold selectivity for JAK1 over JAK2 and for JAK1 over JAK3, respectively [181]. The drug was approved by FDA, and, more recently, by EMA, at a dosage of 15 mg once daily, for adults with moderately to severely active RA who fail to adequately respond or are intolerant to MTX [182]. Like first generation JAKi, it may be prescribed with or without MTX. The efficacy of upadacitinib has been evaluated in two phase II trials (BALANCE 1 and BALANCE 2) [84,183], one phase IIb/III trial (SELECT-SUNRISE) [184] and five phase III RCTs (SELECT-NEXT, SELECT-BEYOND, SELECT-MONOTHERAPY, SELECT-EARLY, SELECT-COMPARE) [185–190]. The phase III trial SELECT-CHOICE, comparing upadacitinib and abatacept in RA patients with an inadequate response to c/bDMARDs is ongoing, and results have not published yet. Results from BALANCE 1 and BALANCE 2 [84,183], enrolling anti-TNF failing and MTX-failing patients respectively, showed rapid, dose-dependent improvements in RA signs and symptoms, with a similar safety and tolerability profile to those of other JAKi. Phase III RCTs recruited patients with inadequate response to at least one cDMARD, including MTX (SELECT-NEXT, SELECT-MONOTHERAPY and SELECT-COMPARE) [185,187,189], and patients with inadequate response or intolerance to bDMARDs (SELECT-BEYOND) [186]. Patients were randomly assigned to received once-daily extended-release formulations of upadacitinib 15 or 30 mg or placebo for at least 12 weeks. Overall, results of these studies showed a rapid statistically significant improvement in the ACR20 response as early as week 1, and in the ACR50 and ACR70 responses from week 2 onward with upadacitinib 15 and 30 mg [185,186]. DAS28-CRP and CDAI scores were significantly improved with both the two upadacitinib doses, with 40–50% of patients achieving low disease activity by week 12. Quality of life, physical function, fatigue, severity, and duration of morning stiffness were also significantly improved in upadacitinib arms regardless of the dose. Of note, in the SELECT-BEYOND trial, recruiting 498 RA patients failing previous lines with biologics (anti-TNF and anti-IL-6R agents), the efficacy outcomes were achieved in the upadacitinib arm vs. placebo regardless of the number or kind of previously received treatments [186]. In the SELECT-MONOTHERAPY phase III RCT [187], upadacitinib monotherapy led to statistically significant improvements in clinical and functional outcomes vs. the continuation of MTX in MTX-resistant patients. A higher and statistically significant proportion of patients receiving both the two upadacitinib doses achieved DAS28-CRP low disease activity or remission compared to those assigned to MTX alone. In the SELECT-COMPARE trial, upadacitinib, at a dose of 15 mg once daily, outperformed adalimumab in the achievement of ACR50, HAQ and DAS28-CRP responses at week 12 in MTX-refractory RA patients. Furthermore, a higher percentage of upadacitinib-assigned patients were in low disease activity or remission at week 26 compared to the adalimumab arm [189,191]. The safety profile of upadacitinib was in line with that of non-selective JAKi. Adverse events in BALANCE 1 and 2 increased in a dose-dependent manner but were mostly mild and included infections (the most common adverse events), nausea, headache, transient increase in serum transaminases and in lipid levels (both LDL and HDL with unchanged ratio) [84,183]. A dose-dependent decrease in the levels of hemoglobin (grade 3 and 4 anemia) was also noted, as well as a decrease in lymphocyte, NK cell and neutrophil count. Serious infections occurred in the upadacitinib 30 mg arms of the SELECT-NEXT and SELECT-BEYOND trials, but none had a fatal course [185,186]. An increased incidence of HZV reactivation was observed in all the upadacitinib treatment arms across the five trials, with two serious cases in the upadacitinib 30 mg group in the SELECT-BEYOND study [186]. Two malignancies and one major MACE occurred in the upadacitinib 30 mg arm of the SELECT-NEXT trial [185], whereas four Biomolecules 2020, 10, 1002 25 of 40 malignancies and two MACEs were reported in the SELECT-BEYOND trial in upadacitinib arms [186]. Four cases of pulmonary embolism were also reported in the SELECT-BEYOND study, but all the patients had known additional risk factors. In the SELECT-COMPARE trial an increase in serum creatine phosphokinase (CPK) was reported in subjects receiving upadacitinib but not in those treated with the active comparator [191]. Upadacitinib is metabolized by CYP enzymes, including CYP3A, but it can be safely taken with other CYP3A-metabolized drugs, including [192], which might be co-prescribed due to the paradoxical effect of JAKi on lipid transport. However, attention must be paid to the plausible synergistic effect of upadacitinib and statins in inducing CPK elevation and skeletal muscle damage.

6.2. Peficitinib Peficitinib (IUPAC name: 4-[[(1R,3S)-5-hydroxy-2-adamantyl]amino]-1H-pyrrolo[2,3-b]pyridine-5- carboxamide) is an oral JAKi approved in March 2019 in Japan for the treatment of RA in patients who have an inadequate response to conventional therapies [193]. The drug is still under pending development in the US and in Europe. The recommended dose of peficitinib is 150 mg or less, depending on patient’s condition, in an after meal single daily administration [194]. Peficitinib displays a less target selectivity than other second generation JAKi. Together with tofacitinib, it is, in fact, considered a pan-JAK inhibitor with a higher selectivity for JAK3 (IC50 = 710 nmol/mL) than for the other JAKs [94]. The low selectivity for JAK2 limits its effects on both red blood cells and platelets suggesting a good safety profile [195]. Findings from phase I studies on healthy volunteers demonstrated that peficitinib is rapidly absorbed in fasting condition. Tmax ranged from 1 h to 1.8 h, depending on the dose, and pharmacokinetics was not significantly influenced by renal impairment [196]. Meals, instead, delay median Tmax from 1.5 h to 4.0 h and increase peficitinib exposure (AUC) by 27% [197]. In addition, the high elimination half-life allows the administration of the drug once a day [198]. Two phase II studies, involving Japanese and non-Japanese patients, aimed to investigate the efficacy and safety of peficitinib in RA patients [198,199]. The studies showed a statistically significant improvement in the ACR20 response rate at week 12 and a similar number of adverse events compared with placebo. A third study, conducted in non-Japanese (North and Latin Americans) RA patients receiving peficitinib in combination with MTX, did not reach the prefixed endpoints due to a high rate of response in the placebo group [200]. The approval of peficitinib was based mainly on the results of two 52 week phase III studies (RAJ3 and RAJ4) [201,202], aiming to evaluate the efficacy and safety of peficitinib, at a single daily dose of 100 mg or 150 mg alone or in combination with cDMARDs, in RA patients non-responder to conventional therapies. The superiority of peficitinib over placebo was demonstrated in both RAJ3 and RAJ4 studies. At week 12, peficitinib clearly outperformed placebo in terms of ACR20 and ACR50 response rates in the RAJ3 study [201], while the ACR70 response was significantly achieved only in the 150 mg peficitinib arm. In the RAJ4 trial, a significant change from baseline in mTSS at week 28 was also reported [202]. The safety profile was in line with that of other already available JAKi and no red flags presented during these studies.

6.3. Filgotinib Filgotinib (IUPAC name: N-[5-[4-[(1,1-dioxo-1,4-thiazinan-4-yl)methyl]phenyl]-[1,2,4]triazolo [1,5-a]pyridin-2-yl]cyclopropanecarboxamide) was designed following a kinase-focused-high-throughput library screening, which identified triazolopyridines as JAK-1 selective catalytic inhibitors [203]. The drug, currently under regulatory review, potently and selectively inhibits JAK1 (IC50 = 629 nmol/mL) [94]. Due to its target selectivity, filgotinib is estimated to have a good efficacy and a better safety profile than unselective JAKi. In particular, as JAK1 is not involved in the signaling pathway of Biomolecules 2020, 10, 1002 26 of 40 erythropoietin, colony-stimulating factor, and thrombopoietin, filgotinib should not increase the risk of anemia and thrombocytopenia [204]. The efficacy and safety of filgotinib in MTX-unresponsive RA patients were tested in two exploratory phase IIa trials [205], including a monocentric four-week proof of concept study and a multicentric four-week preliminary study. In the first trial, patients were randomized to receive filgotinib 200 mg daily or placebo, whereas in the second one, they were randomly assigned to different doses of filgotinib (30 mg, 75 mg, 150 mg, or 300 mg once daily) or placebo. Both the studies evidenced a satisfactory efficacy profile of the drug. In the proof of concept study, a statistically significant number of patients achieved an ACR20 response compared to placebo. Furthermore, a reduction in serum CRP levels and in DAS28-CRP scores was also reported in the filgotinib-assigned arm. Conversely, the treatment with filgotinib was not associated with a significant improvement in the ACR20 response rate in the four-week preliminary study, although a trend was observed for the 300 mg dose. No safety issues were reported. These initial data together with those obtained from healthy volunteers paved the way for the development of a population’s pharmacokinetic/pharmacodynamic model to be used for dose selection in phase IIb studies [206,207]. The pharmacodynamic effect of filgotinib is, in fact, given by the parent drug and its active metabolite. The latter derives from the loss of the cyclopropyl carboxylic acid group following the action of [204]. Despite having a lower target selectivity for JAK1 compared to filgotinib (IC50 = 11.9 µmol/ml), the active metabolite has an elimination half-life of 23 h, allowing the administration of the compound in a single daily dose [205]. A wide dose range and different dosing regimens, with or without MTX, were investigated in 24-week dose finding phase IIb studies in patients with active RA despite the concomitant use of MTX (DARWIN 1 and DARWIN 2) [206,207]. Results showed that filgotinib at a dose of 100 or 200 mg once daily, given either as mono- or combo-therapy, was efficacious and well tolerated. Recently, the published results of the 24 week FINCH2 phase III study confirmed the efficacy of filgotinib 100 mg and 200 mg once daily in RA patients refractory to one or more bDMARD [208]. No opportunistic infections, malignancies, or fatalities were recorded during the observational period.

6.4. Decernotinib Decernotinib (IUPAC name: (2R)-2-methyl-2-[[2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl] amino]-N-(2,2,2-trifluoroethyl)butanamide) is an oral selective JAK3 inhibitor whose development for the treatment of RA is presumed to have been discontinued. JAK3 is expressed in lymphoid cells and its blockade may prevent the signaling of several cytokines involved in autoimmunity, including IL-2, IL-4, IL-7, and IL-15 [209], without affecting red blood cells and platelets. Decernotinib was discovered following a library screening of compounds targeting JAK3 and chemically modified in order to enhance its binding-affinity and potency [210]. The drug was tested in phase IIa and IIb RCTs as monotherapy [211] or in combination with MTX [212] or other cDMARDs [213]. When given as monotherapy in RA patients with uncontrolled disease despite the use of at least 1 cDMARD, decernotinib, at dosages of 25 mg, 50 mg, 100 mg, or 150 mg twice a day, led to a significant improvement in the ACR20 response and DAS28-CRP scores at week 12 compared to placebo. Best benefits were observed in patients who received one of the 3 higher doses (50 mg, 100 mg, and 150 mg). Clinical remission, defined by a DAS28-CRP score < 2.6, was significantly greater in patients assigned to decernotinib 100 mg and 150 mg compared to placebo. The administration of decernotinib at a dose of 150 mg twice a day also resulted in better ACR50 and ACR70 response rates than those observed with placebo. Dose titration studies showed that the ACR50 response was significantly higher with all the doses at week 12 and 24, while the achievement of the ACR70 response at week 12 was only significant with the doses of 150 mg once a day and of 100 mg twice a day [212]. Genovese et al. investigated the efficacy of the drug in the achievement of clinical and magnetic resonance imaging (MRI) outcomes in patients with inadequate DMARD response and randomly Biomolecules 2020, 10, 1002 27 of 40 assigned to decernotinib (100 mg, 200 mg or 300 mg once daily) or placebo for 12 weeks [213]. Compared to placebo, the ACR20, DAS28-CRP and RA MRI scoring (RAMRIS) responses improved over 12 weeks across all the decernotinib dosages in a dose-dependent manner when combined with a stable cDMARD background. The treatment with decernotinib was generally well tolerated. Adverse events were more frequent when the drug was administered at higher doses. The most common adverse events were nausea, headache, HZV reactivation, nasopharyngitis, diarrhea, upper respiratory trait infections, increased ALT serum levels, and hypercholesterolemia. In the phase IIa RCT, the incidence of infections was similar between placebo and decernotinib assigned groups, but the frequency was higher with the 100 and 150 mg doses compared to 50 mg and 25 mg doses. Five serious infections occurred, all in the decernotinib treatment arms, with two fatalities due to pneumonia and subarachnoid hemorrhage. Twelve of the 13 serious infections reported in the phase IIb studies (decernotinib + MTX) occurred in decernotinib-treated patients (with nine cases of pneumonia/bronchitis), and included two fatalities due to pneumonia and cardiac failure. Other adverse events encompassed increases in serum lipids and creatinine and the decrease in lymphocyte blood count, all of which were dose-dependent. As decernotinib is a potent inhibitor of CYP3A4, this may represent a limitation in its use when coprescribed with CYP3A4-metabolized medications, like statins.

6.5. Itacitinib Itacitinib (INCB039110; Corporation, Wilmington, DE) (IUPAC name: 2-[1-[1-[3-fluoro-2-(trifluoromethyl)pyridine-4-carbonyl]piperid in-4-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimi din-4-yl)pyrazol-1-yl]azetidin-3-yl]acetonitrile) is a novel oral selective JAK1 inhibitor, approved in 2018 by EMA as an orphan drug for the treatment of graft-versus-host disease (GVHD) [214], and currently tested for other clinical indications, including oncologic and immunologic disorders. In RA, itacitinib has been tested in a phase II randomized, dose-ranging, placebo-controlled trial [215]. The study evaluated the efficacy of itacitinib 100 mg twice a day, 200 mg twice a day, and 300 and 600 mg once daily vs. placebo in patients with active RA despite cDMARDs. The study showed that patients assigned to the highest dose had better clinical improvements and a sustained ACR20 response than patients in the other arms, regardless of background therapy or previous biologic experience. Responses were observed as early as at the first assessment time (14 days). Itacitinib was generally well tolerated. No grade 3 or grade 4 adverse events (AE) nor serious and opportunistic infections were reported. A dose-related increase in LDL was noted, though without any change in the HDL/LDL ratio. Safety data obtained from studies on GVHD patients treated with itacitinib suggest that no dose adjustment is required for a single dose of 300 mg even in patients with severe renal impairment or end-stage renal disease [216]. Furthermore, studies on healthy participants showed that single doses ranging from 10 to 1000 mg are safe from a cardiovascular point of view [217]. Nevertheless, to date, no registration trials have been designed for the use of itacitinib in RA.

7. Concluding Discussion The JAK-STAT signaling pathway plays a central role in many physiopathological processes, including antimicrobial defense, hematopoiesis, post-natal growth and metabolism [71]. When activated in a canonical way, it mediates cytokine and growth factor intracellular signals; however, a non-canonical activation has also been described and associated with the epigenetic control of chromatin stability and with cell homeostasis [74]. Consequently, JAK-STAT dysregulation may be at the basis of many pathological conditions, ranging from solid and hematologic malignancies to resistance, and immune-mediated diseases [71,93,146,218,219]. Hence, several JAKi have been developed for the treatment of myeloproliferative and lymphoproliferative disorders, as well as for solid tumors [71]. More recently, the advent of the anti-rheumatic JAKi constituted a breakthrough in the therapeutic algorithm of RA and other inflammatory diseases, including spondyloarthritis and inflammatory Biomolecules 2020, 10, 1002 28 of 40 bowel diseases. The panorama of JAKi is constantly enriching with new incoming molecules designed to be more specific on targeted cells and pathways, thus resulting in a better efficacy and safety profile. Compared to their biologic counterparts, JAKi differ in the route of administration, safety and efficacy profile and costs of manufacturing. Given the evidence of superiority or non-inferiority of JAKi vs. adalimumab emerging from RCTs [125,156,190], the 2020 updated EULAR therapeutic guidelines [113] recommended the use of JAKi as an alternative to biologics in RA patients refractory to cDMARDs and having poor prognostic factors, as well as in those failing a previous biologic or synthetic line [24]. A preference for biologics or JAKi should be accorded based on contraindications, monotherapy need or cost issues. Furthermore, data from RCTs evidenced a rapid and maintained efficacy of JAKi regardless of the co-prescription of MTX or other cDMARDs, and the safety profile of JAKi appeared in line with that of other authorized treatments for RA. However, some side effects, including thromboembolic events or HZV reactivation, seem to specifically occur with this class of medications. Due to a higher target selectivity, second generation JAKi may display a better safety profile. Nevertheless, several questions remain unanswered and should be inscribed in future research agendas: (1) the identification of biomarkers (e.g., ACPA or RF positivity) predicting a better response to JAKi; (2) the duration of JAKi treatment and issues related to a dose-reduction strategy or discontinuation in patients being in low disease activity or remission; (3) the interference played, in the long term, by other CYP-metabolized drugs on JAKi pharmacokinetic profile; (4) the efficacy and safety derived from a JAKi cycling strategy and from the combination of JAKi with bDMARDs; (5) the efficacy and safety of JAKi compared to non-anti-TNF biologics. Indirect comparison data from network meta-analysis and patient cohort registries have partly addressed the latter point with regard to tofacitinib [160–165], but no evidence currently exists for other JAKi. Growing experience on already marketed JAKi and cumulative experimental findings on novel compounds are expected to clarify many of these aspects in the next years.

Author Contributions: G.B., G.F., J.A., L.D.C., R.R., and S.B. drafted the original manuscript and tables, performed the bibliographic research and drew the figures. R.T. drew the figures, helped in writing and implemented the manuscript performing a second literature research. J.A., R.T. and F.S. critically reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare that they do not have any conflict of interests.

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